Añadir Con qué perspectivas de aquí atraviesan a la tesis. Merleau Ponty, pero también las reflexiones de artistas que trabajan sobre este mismo tema.

moet mannen zijn

eLife Assessment

This valuable study explores the role of the chromatin regulator ATAD2 in mouse spermatogenesis. The data convincingly demonstrate that ATAD2 is essential for proper chromatin remodeling in haploid spermatids, influencing gene accessibility, H3.3-mediated transcription, and histone eviction. Using Atad2 knockout (KO) mice, the authors link ATAD2 to the DNA-replication-independent incorporation of sperm-specific proteins like protamines and histone H3.3. Although the findings highlight chromatin abnormalities and impaired in vitro fertilization in KO mice, natural fertility remains unaffected, suggesting possible in vivo compensatory mechanisms. Future experiments will be needed to tease out the precise molecular role of ATAD2 in spermatogenesis. This work will be of interest to the epigenetics and developmental fields.

Reviewer #1 (Public review):

Summary:

The authors analyzed the expression of ATAD2 protein in post-meiotic stages and characterized the localization of various testis-specific proteins in the testis of the Atad2 knockout (KO). By cytological analysis as well as the ATAC sequencing, the study showed that increased levels of HIRA histone chaperone, accumulation of histone H3.3 on post-meiotic nuclei, defective chromatin accessibility and also delayed deposition of protamines. Sperm from the Atad2 KO mice reduces the success of in vitro fertilization. The work was performed well, and most of the results are convincing. However, this manuscript does not suggest a molecular mechanism for how ATAD2 promotes the formation of testis-specific chromatin.

Strengths:

The paper describes the role of ATAD2 AAA+ ATPase in the proper localization of sperm-specific chromatin proteins such as protamine, suggesting the importance of the DNA replication-independent histone exchanges with the HIRA-histone H3.3 axis.

Weaknesses:

The work was performed well, and most of the results are convincing. However, this manuscript does not suggest a molecular mechanism for how ATAD2 promotes the formation of testis-specific chromatin.

Reviewer #2 (Public review):

Summary:

This manuscript by Liakopoulou et al. presents a comprehensive investigation into the role of ATAD2 in regulating chromatin dynamics during spermatogenesis. The authors elegantly demonstrate that ATAD2, via its control of histone chaperone HIRA turnover, ensures proper H3.3 localization, chromatin accessibility, and histone-to-protamine transition in post-meiotic male germ cells. Using a new well-characterized Atad2 KO mouse model, they show that ATAD2 deficiency disrupts HIRA dynamics, leading to aberrant H3.3 deposition, impaired transcriptional regulation, delayed protamine assembly, and defective sperm genome compaction. The study bridges ATAD2's conserved functions in embryonic stem cells and cancer to spermatogenesis, revealing a novel layer of epigenetic regulation critical for male fertility.

Strengths:

The MS first demonstration of ATAD2's essential role in spermatogenesis, linking its expression in haploid spermatids to histone chaperone regulation by connecting ATAD2-dependent chromatin dynamics to gene accessibility (ATAC-seq), H3.3-mediated transcription, and histone eviction. Interestingly and surprisingly, sperm chromatin defects in Atad2 KO mice impair only in vitro fertilization but not natural fertility, suggesting unknown compensatory mechanisms in vivo.

Weaknesses:

The MS is robust and there are not big weaknesses

The authors have addressed all the queries successfully.

Reviewer #3 (Public review):

Summary:

The authors generated knockout mice for Atad2, a conserved bromodomain-containing factor expressed during spermatogenesis. In Atad2 KO mice, HIRA, a chaperone for histone variant H3.3, was upregulated in round spermatids, accompanied by an apparent increase in H3.3 levels. Furthermore, the sequential incorporation and removal of TH2B and PRM1 during spermiogenesis were partially disrupted in the absence of ATAD2, possibly due to delayed histone removal. Despite these abnormalities, Atad2 KO male mice were able to produce offspring normally.

Strengths:

The manuscript addresses the biological role of ATAD2 in spermatogenesis using a knockout mouse model, providing a valuable in vivo framework to study chromatin regulation during male germ cell development. The observed redistribution of H3.3 in round spermatids is clearly presented and suggests a previously unappreciated role of ATAD2 in histone variant dynamics. The authors also document defects in the sequential incorporation and removal of TH2B and PRM1 during spermiogenesis, providing phenotypic insight into chromatin transitions in late spermatogenic stages. Overall, the study presents a solid foundation for further mechanistic investigation into ATAD2 function.

Weaknesses:

While the manuscript reports the gross phenotype of Atad2 KO mice, the findings remain largely superficial and do not convincingly demonstrate how ATAD2 deficiency affects chromatin.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review): 

Summary: 

The authors analyzed the expression of ATAD2 protein in post-meiotic stages and characterized the localization of various testis-specific proteins in the testis of the Atad2 knockout (KO). By cytological analysis as well as the ATAC sequencing, the study showed that increased levels of HIRA histone chaperone, accumulation of histone H3.3 on post-meiotic nuclei, defective chromatin accessibility and also delayed deposition of protamines. Sperm from the Atad2 KO mice reduces the success of in vitro fertilization. The work was performed well, and most of the results are convincing. However, this manuscript does not suggest a molecular mechanism for how ATAD2 promotes the formation of testis-specific chromatin. 

We would like to take this opportunity to highlight that the present study builds on our previously published work, which examined the function of ATAD2 in both yeast S. pombe and mouse embryonic stem (ES) cells (Wang et al., 2021). In yeast, using genetic analysis we showed that inactivation of HIRA rescues defective cell growth caused by the absence of ATAD2. This rescue could also be achieved by reducing histone dosage, indicating that the toxicity depends on histone over-dosage, and that HIRA toxicity, in the absence of ATAD2, is linked to this imbalance.

Furthermore, HIRA ChIP-seq performed in mouse ES cells revealed increased nucleosome-bound HIRA, particularly around transcription start sites (TSS) of active genes, along with the appearance of HIRA-bound nucleosomes within normally nucleosome-free regions (NFRs). These findings pointed to ATAD2 as a major factor responsible for unloading HIRA from nucleosomes. This unloading function may also apply to other histone chaperones, such as FACT (see Wang et al., 2021, Fig. 4C).

In the present study, our investigations converge on the same ATAD2 function in the context of a physiologically integrated mammalian system—spermatogenesis. Indeed, in the absence of ATAD2, we observed H3.3 accumulation and enhanced H3.3-mediated gene expression. Consistent with this functional model of ATAD2— unloading chaperones from histone- and non-histone-bound chromatin—we also observed defects in histone-toprotamine replacement.

Together, the results presented here and in Wang et al. (2021) reveal an underappreciated regulatory layer of histone chaperone activity. Previously, histone chaperones were primarily understood as factors that load histones. Our findings demonstrate that we must also consider a previously unrecognized regulatory mechanism that controls assembled histone-bound chaperones. This key point was clearly captured and emphasized by Reviewer #2 (see below).

Strengths:

The paper describes the role of ATAD2 AAA+ ATPase in the proper localization of sperm-specific chromatin proteins such as protamine, suggesting the importance of the DNA replication-independent histone exchanges with the HIRA-histone H3.3 axis. 

Weaknesses: 

(1) Some results lack quantification. 

We will consider all the data and add appropriate quantifications where necessary.

(2) The work was performed well, and most of the results are convincing. However, this manuscript does not suggest a molecular mechanism for how ATAD2 promotes the formation of testis-specific chromatin. 

Please see our comments above.

Reviewer #2 (Public review): 

Summary:

This manuscript by Liakopoulou et al. presents a comprehensive investigation into the role of ATAD2 in regulating chromatin dynamics during spermatogenesis. The authors elegantly demonstrate that ATAD2, via its control of histone chaperone HIRA turnover, ensures proper H3.3 localization, chromatin accessibility, and histone-toprotamine transition in post-meiotic male germ cells. Using a new well-characterized Atad2 KO mouse model, they show that ATAD2 deficiency disrupts HIRA dynamics, leading to aberrant H3.3 deposition, impaired transcriptional regulation, delayed protamine assembly, and defective sperm genome compaction. The study bridges ATAD2's conserved functions in embryonic stem cells and cancer to spermatogenesis, revealing a novel layer of epigenetic regulation critical for male fertility. 

Strengths:

The MS first demonstration of ATAD2's essential role in spermatogenesis, linking its expression in haploid spermatids to histone chaperone regulation by connecting ATAD2-dependent chromatin dynamics to gene accessibility (ATAC-seq), H3.3-mediated transcription, and histone eviction. Interestingly and surprisingly, sperm chromatin defects in Atad2 KO mice impair only in vitro fertilization but not natural fertility, suggesting unknown compensatory mechanisms in vivo. 

Weaknesses:

The MS is robust and there are not big weaknesses 

Reviewer #3 (Public review): 

Summary: 

The authors generated knockout mice for Atad2, a conserved bromodomain-containing factor expressed during spermatogenesis. In Atad2 KO mice, HIRA, a chaperone for histone variant H3.3, was upregulated in round spermatids, accompanied by an apparent increase in H3.3 levels. Furthermore, the sequential incorporation and removal of TH2B and PRM1 during spermiogenesis were partially disrupted in the absence of ATAD2, possibly due to delayed histone removal. Despite these abnormalities, Atad2 KO male mice were able to produce offspring normally. 

Strengths:

The manuscript addresses the biological role of ATAD2 in spermatogenesis using a knockout mouse model, providing a valuable in vivo framework to study chromatin regulation during male germ cell development. The observed redistribution of H3.3 in round spermatids is clearly presented and suggests a previously unappreciated role of ATAD2 in histone variant dynamics. The authors also document defects in the sequential incorporation and removal of TH2B and PRM1 during spermiogenesis, providing phenotypic insight into chromatin transitions in late spermatogenic stages. Overall, the study presents a solid foundation for further mechanistic investigation into ATAD2 function. 

Weaknesses:

While the manuscript reports the gross phenotype of Atad2 KO mice, the findings remain largely superficial and do not convincingly demonstrate how ATAD2 deficiency affects chromatin dynamics. Moreover, the phenotype appears too mild to elucidate the functional significance of ATAD2 during spermatogenesis. 

We respectfully disagree with the statement that our findings are largely superficial. Based on our investigations of this factor over the years, it has become evident that ATAD2 functions as an auxiliary factor that facilitates mechanisms controlling chromatin dynamics (see, for example, Morozumi et al., 2015). These mechanisms can still occur in the absence of ATAD2, but with reduced efficiency, which explains the mild phenotype we observed.

This function, while not essential, is nonetheless an integral part of the cell’s molecular biology and should be studied and brought to the attention of the broader biological community, just as we study essential factors. Unfortunately, the field has tended to focus primarily on core functional actors, often overlooking auxiliary factors. As a result, our decade-long investigations into the subtle yet important roles of ATAD2 have repeatedly been met with skepticism regarding its functional significance, which has in turn influenced editorial decisions.

We chose eLife as the venue for this work specifically to avoid such editorial barriers and to emphasize that facilitators of essential functions do exist. They deserve to be investigated, and the underlying molecular regulatory mechanisms must be understood.

(1) Figures 4-5: The analyses of differential gene expression and chromatin organization should be more comprehensive. First, Venn diagrams comparing the sets of significantly differentially expressed genes between this study and previous work should be shown for each developmental stage. Second, given the established role of H3.3 in MSCI, the effect of Atad2 knockout on sex chromosome gene expression should be analyzed. Third, integrated analysis of RNA-seq and ATAC-seq data is needed to evaluate how ATAD2 loss affects gene expression. Finally, H3.3 ChIP-seq should be performed to directly assess changes in H3.3 distribution following Atad2 knockout.  

(1) In the revised version, we will include Venn diagrams to illustrate the overlap in significantly differentially expressed genes between this study and previous work. However, we believe that the GSEAs presented here provide stronger evidence, as they indicate the statistical significance of this overlap (p-values). In our case, we observed p-value < 0.01 (**) and p < 0.001 (***).

(2) Sex chromosome gene expression was analyzed and is presented in Fig. 5C.

(3) The effect of ATAD2 loss on gene expression is shown in Fig. 4A, B, and C as histograms, with statistical significance indicated in the middle panels.

(4) Although mapping H3.3 incorporation across the genome in wild-type and Atad2 KO cells would have been informative, the available anti-H3.3 antibody did not work for ChIP-seq, at least in our hands. The authors of Fontaine et al., 2022, who studied H3.3 during spermatogenesis in mice, must have encountered the same problem, since they tagged the endogenous H3.3 gene to perform their ChIP experiments.

(2) Figure 3: The altered distribution of H3.3 is compelling. This raises the possibility that histone marks associated with H3.3 may also be affected, although this has not been investigated. It would therefore be important to examine the distribution of histone modifications typically associated with H3.3. If any alterations are observed, ChIP-seq analyses should be performed to explore them further.

Based on our understanding of ATAD2’s function—specifically its role in releasing chromatin-bound HIRA—in the absence of ATAD2 the residence time of both HIRA and H3.3 on chromatin increases. This results in the detection of H3.3 not only on sex chromosomes but across the genome. Our data provide clear evidence of this phenomenon. The reviewer is correct in suggesting that the accumulated H3.3 would carry H3.3-associated histone PTMs; however, we are unsure what additional insights could be gained by further demonstrating this point.

(3) Figure 7: While the authors suggest that pre-PRM2 processing is impaired in Atad2 KO, no direct evidence is provided. It is essential to conduct acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by western blotting, or a comparable experiment, to substantiate this claim. 

Figure 7 does not suggest that pre-PRM2 processing is affected in Atad2 KO; rather, this figure—particularly Fig. 7B—specifically demonstrates that pre-PRM2 processing is impaired, as shown using an antibody that recognizes the processed portion of pre-PRM2. ELISA was used to provide a more quantitative assessment; however, in the revised manuscript we will also include a western blot image.

(4) HIRA and ATAD2: Does the upregulation of HIRA fully account for the phenotypes observed in Atad2 KO? If so, would overexpression of HIRA alone be sufficient to phenocopy the Atad2 KO phenotype? Alternatively, would partial reduction of HIRA (e.g., through heterozygous deletion) in the Atad2 KO background be sufficient to rescue the phenotype? 

These are interesting experiments that require the creation of appropriate mouse models, which are not currently available.

(5) The mechanism by which ATAD2 regulates HIRA turnover on chromatin and the deposition of H3.3 remains unclear from the manuscript and warrants further investigation. 

The Reviewer is absolutely correct. In addition to the points addressed in response to Reviewer #1’s general comments (see above), it would indeed have been very interesting to test the segregase activity of ATAD2 (likely driven by its AAA ATPase activity) through in vitro experiments using the Xenopus egg extract system described by Tagami et al., 2004. This system can be applied both in the presence and absence (via immunodepletion) of ATAD2 and would also allow the use of ATAD2 mutants, particularly those with inactive AAA ATPase or bromodomains. However, such experiments go well beyond the scope of this study, which focuses on the role of ATAD2 in chromatin dynamics during spermatogenesis.

References:

(1) Wang T, Perazza D, Boussouar F, Cattaneo M, Bougdour A, Chuffart F, Barral S, Vargas A, Liakopoulou A, Puthier D, Bargier L, Morozumi Y, Jamshidikia M, Garcia-Saez I, Petosa C, Rousseaux S, Verdel A, Khochbin S. ATAD2 controls chromatin-bound HIRA turnover. Life Sci Alliance. 2021 Sep 27;4(12):e202101151. doi: 10.26508/lsa.202101151. PMID: 34580178; PMCID: PMC8500222.

(2) Morozumi Y, Boussouar F, Tan M, Chaikuad A, Jamshidikia M, Colak G, He H, Nie L, Petosa C, de Dieuleveult M, Curtet S, Vitte AL, Rabatel C, Debernardi A, Cosset FL, Verhoeyen E, Emadali A, Schweifer N, Gianni D, Gut M, Guardiola P, Rousseaux S, Gérard M, Knapp S, Zhao Y, Khochbin S. Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells. J Mol Cell Biol. 2016 Aug;8(4):349-62. doi: 10.1093/jmcb/mjv060. Epub 2015 Oct 12. PMID: 26459632; PMCID: PMC4991664.

(3) Fontaine E, Papin C, Martinez G, Le Gras S, Nahed RA, Héry P, Buchou T, Ouararhni K, Favier B, Gautier T, Sabir JSM, Gerard M, Bednar J, Arnoult C, Dimitrov S, Hamiche A. Dual role of histone variant H3.3B in spermatogenesis: positive regulation of piRNA transcription and implication in X-chromosome inactivation. Nucleic Acids Res. 2022 Jul 22;50(13):7350-7366. doi: 10.1093/nar/gkac541. PMID: 35766398; PMCID: PMC9303386.

(4) Tagami H, Ray-Gallet D, Almouzni G, Nakatani Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell. 2004 Jan 9;116(1):51-61. doi: 10.1016/s0092-8674(03)01064-x. PMID: 14718166.

Recommendations for the authors:

Reviewing Editor Comments:

I note that the reviewers had mixed opinions about the strength of the evidence in the manuscript. A revision that addresses these points would be welcome.

Reviewer #1 (Recommendations for the authors):  

Major points: 

(1) No line numbers: It is hard to point out the issues.

The revised version harbors line numbers.

(2) Given the results shown in Figure 3 and Figure 4, it is nice to show the chromosomal localization of histone H3.3 in spermatocytes or post-meiotic cells by Chromatin-immunoprecipitation sequencing (ChIP-seq).

Although mapping H3.3 incorporation across the genome in wild-type and Atad2 KO cells would have been informative, the available anti-H3.3 antibody did not work for ChIP-seq in our hands. In fact, this antibody is not well regarded for ChIP-seq. For example, Fontaine et al. (2022), who investigated H3.3 during spermatogenesis in mice, circumvented this issue by tagging the endogenous H3.3 genes for their ChIP experiments.

(3) Figure 7B and 8: Why the authors used ELISA for the protein quantification. At least, western blotting should be shown.

ELISA is a more quantitative method than traditional immunoblotting. Nevertheless, as requested by the reviewer, we have now included a corresponding western blot in Fig. S3.

(4) For readers, please add a schematic pathway of histone-protamine replacement in sperm formation in Fig.1 and it would be nice to have a model figure, which contains the authors' idea in the last figure.

As requested by this reviewer, we have now included a schematic model in Figure 9 to summarize the main conclusions of our work.

Minor points: 

(1) Page 2, the second paragraph, "pre-PRM2: Please explain more about pre-PRM2 and/or PRM2 as well as PRM1 (Figure 6).

More detailed descriptions of PRM2 processing are now given in this paragraph. 

(2) Page 3, bottom paragraph, line 1: "KO" should be "knockout (KO)".

Done.

(3) Page 4, second paragraph bottom: Please explain more about the protein structure of germ-line-specific ATAD2S: how it is different from ATAD2L. Germ-line specific means it is also expressed in ovary?

As Atad2 is predominantly expressed in embryonic stem cells and in spermatogenic cells, we replaced all through the text germ-line specific by more appropriate terms.

(4) Figure 1C, western blotting: Wild-type testis extracts, both ATAD2L and -S are present. Does this mean that ATADS2L is expressed in both germ line as well as supporting cells. Please clarify this and, if possible, show the western blotting of spermatids well as spermatocytes.

Figure 1D shows sections of seminiferous tubules from Atad2 KO mice, in which lacZ expression is driven by the endogenous Atad2 promoter. The results indicate that Atad2 is expressed mainly in post-meiotic cells. Most labeled cells are located near the lumen, whereas the supporting Sertoli cells remain unlabeled. Sertoli cells, which are anchored to the basal lamina, span the entire thickness of the germinal epithelium from the basal lamina to the lumen. Their nuclei, however, are usually positioned closer to the basal membrane. Thus, the observed lacZ expression pattern argues against substantial Atad2 expression in Sertoli cells. 

(5) Figure 1C: Please explain a bit more about the reduction of ATAD2 proteins in heterozygous mice.

Done

(6) Figure 1C: Genotypes of the mice should be shown in the legend.

Done 

(7) Figure 1D: Please add a more magnified image of the sections to see the staining pattern in the seminiferous tubules.

The magnification does not bring more information since we lose the structure of cells within tubules due the nature of treatment of the sections for X-gal staining. Please see comments to question 1C to reviewer 2

(8) Page 5, first paragraph, line 2, histone dosage: What do the authors meant by the histone dosage? Please explain more or use more appropriate word.

"Histone dosage" refers to the amount or relative abundance of histone proteins in a cell.

(9) Figure 2A: Figure 2A: Given the result in Figure 1C, it is interesting to check the amount of HIRA in Atad2 heterozygous mice.

In Atad2 heterozygous mice, we would expect an increase in HIRA, but only to about half the level seen in the Atad2 homozygous knockout shown in Figure 2A, which is relatively modest. Therefore, we doubt that detecting such a small change—approximately half of that in Figure 2A—would yield clear or definitive results. 

(10) Figure 2A, legend (n=5): What does this "n" mean? The extract of testes from "5" male mice like Figure 2B. Or 5 independent experiments. If the latter is true, it is important to share the other results in the Supplements.

“n” refers to five WT and five Atad2 KO males. The legend has been clarified as suggested by the reviewer.

(11) Figure 2A, legend, line 2, Atad2: This should be italicized.

Done

(12) Figure 2B: Please show the quantification of amounts of HIRA protein like Fig. 2A.

As indicated in the legend, what is shown is a pool of testes from 3 individuals per genotype.

(13) Figure 2B shows an increased level of HIRA in Atad2 KO testis. This suggests the role of ATAD2 in the protein degradation of HIRA. This possibility should be mentioned or tested since ATAD2 is an AAA+ ATPase. 

The extensive literature on ATAD2 provides no indication that it is involved in protein degradation. In our early work on ATAD2 in the 2000s, we hypothesized that, as a member of the AAA ATPase family, ATAD2 might associate with the 19S proteasome subunit (through multimerization with the other AAA ATPase member of this regulatory subunit). However, both our published pilot studies (Caron et al., PMID: 20581866) and subsequent unpublished work ruled out this possibility. Instead, since the amount of nucleosome-bound HIRA increases in the absence of ATAD2, we propose that chromatin-bound HIRA is more stable than soluble HIRA once it has been released from chromatin by ATAD2.

(14) Page 6, second paragraph, line 5, ko: KO should be capitalized.

Done

(15) Page 6, second paragraph, line 2 from the bottom, chromatin dynamics: Throughout the text, the authors used "chromatin dynamics". However, all the authors analyzed in the current study is the localization of chromatin protein.  So, it is much easier to explain the results by using "chromatin status," etc. In this context, "accessibility" is better. 

We changed the term “chromatin dynamics” into a more precise term according to the context used all through the text.

(16) Figure 3: Please provide the quantification of signals of histone H3.3 in a nucleus or nuclear cytoplasm.

This request is not clear to us since we do not observe any H3.3 signal in the cytoplasm.

(17) Figure 3: As the control of specificity in post-meiotic cells, please show the image and quantification of the H3.3 signals in spermatocyte, for example.

This request is not clear to us. What specificity is meant? 

(18) Figure 3, bottom panels: Please show what the white lines indicate? 

The white lines indicate the limit of cell nucleus and estimated by Hoechst staining. This is now indicated in the legend of the figure. 

(19) Figure 4A: Please explain more about what kind of data is here. Is this wild-type and/or Atad2 KO? The label of the Y-axis should be "mean expression level". What is the standard deviation (SD) here on the X-axis. Moreover, there is only one red open circle, but the number of this class is 5611. All 5611 genes in this group show NO expression. Please explain more.

The plot displays the mean expression levels (y-axis, labeled as "mean expression level") versus the corresponding standard deviations (x-axis), both calculated from three independent biological replicates of isolated round spermatids (Atad2 wild-type and Atad2 KO). The standard deviation reflects the variability of gene expression across biological replicates. Genes were grouped into four categories (grp1: blue, grp2: cyan, grp3: green, grp4: orange) according to the quartile of their mean expression. For grp4, all genes have no detectable expression, resulting in a mean expression of zero and a standard deviation of zero; consequently, the 5611 genes in this group are represented by a single overlapping point (red open circle) at the origin. 

(20) Figure 4C: If possible, it would be better to have a statistical comparison between wild-type and the KO.  

The mean profiles are displayed together with their variability (± 2 s.e.m.) across the four replicates for both ATAD2 WT (blue) and ATAD2 KO (red). For groups 1, 2, and 3, the envelopes of the curves remain clearly separated around the peak, indicating a consistent difference in signal between the two conditions. In contrast, group 4 does not present a strong signal and, accordingly, no marked difference is observed between WT and KO in this group.

(21) Figure 5, GSEA panels: Please explain more about what the GSEA is in the legend.  The legend has been updated as follows:

(A) Expression profiles of post-meiotic H3.3-activated genes. The heatmap (left panel) displays the normalized expression levels of genes identified by Fontaine and colleagues as upregulated in the absence of histone H3.3 (Fontaine et al. 2022) for Atad2 WT (WT) and Atad2 KO (KO) samples at days 20, 22, 24, and 26 PP (D20 to D26). The colour scale represents the z-score of log-transformed DESeq2-normalized counts. The middle panel box plots display, pooled, normalized expression levels, aggregated across replicates and genes, for each condition (WT and KO) and each time point (D20 to D26). Statistical significance between WT and KO conditions was determined using a two-sided t-test, with p-values indicated as follows: * for p-value<0.05, ** for p-value<0.01 and *** for p-value<0.001. The right panel shows the results of gene set enrichment analysis (GSEA), which assesses whether predefined groups of genes show statistically significant differences between conditions. Here, the post-meiotic H3.3-activated genes set, identified by Fontaine et al. (2022), is significantly enriched in Atad2 KO compared with WT samples at day 26 (p < 0.05, FDR < 0.25). Coloured vertical bars indicate the “leading edge” genes (i.e., those contributing most to the enrichment signal), located before the point of maximum enrichment score.  (B) As shown in (A) but for the "post-meiotic H3.3-repressed genes" gene set. (C) As shown in (A) but for the " sex chromosome-linked genes " gene set.

(22) Figure 6. In the KO, the number of green cells is more than red and yellow cells, suggesting the delayed maturation of green (TH2B-positive) cells. It is essential to count the number of each cell and show the quantification.

The green cells correspond to those expressing TH2B but lacking transition proteins (TP) and protamine 1 (Prm1), indicating that they are at earlier stages than elongating–condensing spermatids. Counting these green cells simply reflects the ratio of elongating/condensing spermatids to earlier-stage cells, which varies depending on the field examined. The key point in this experiment is that in wild-type mice, only red cells (elongating/condensing spermatids) and green cells (earlier stages) are observed. By contrast, in Atad2 KO testes, a significant proportion of yellow cells appears, which are never seen in wild-type tissue. The crucial metric here is the percentage of yellow cells relative to the total number of elongating/condensing spermatids (red cells). In wild-type testes, this value is consistently 0%, whereas in Atad2 KO testes it always ranges between 50% and 100% across all fields containing substantial numbers of elongating/condensing spermatids.

(23) Figure 8A: Please show the images of sperm (heads) in the KO mice with or without decompaction.

The requested image is now displayed in Figure S5.

(24) Figure 8C: In the legend, it says n=5. However, there are more than 5 plots on the graph. Please explain the experiment more in detail.

The experiment is now better explained in the legend of this Figure.

Reviewer #2 (Recommendations for the authors): 

While the study is rigorous and well performed, the following minor points could be addressed to strengthen the manuscript: 

Figure 1C should indicate each of the different types of cells present in the sections. It would be of interest to show specifically the different post-meiotic germ cells.

With this type of sample preparation, it is difficult to precisely distinguish the different cell types within the sections. Nevertheless, the staining pattern strongly indicates that most of the intensely stained cells are post-meiotic, situated near the tubule lumens and extending roughly halfway toward the basal membrane.

In the absence of functional ATAD2, the accumulation of HIRA primarily occurs in round spermatids (Fig. 2B). If technically possible, it would be of great interest to show this by IHC of testis section. 

Unfortunately, our antibody did not satisfactorily work in IHC.

The increased of H3.3 signal in Atad2 KO spermatids (Fig. 3) is interpreted because of a reduced turnover. However, alternative explanations (e.g., H3.3 misincorporation or altered chaperone affinity) should not be ruled out. 

The referee is correct that alternative explanations are possible. However, based on our previous work (Wang et al., 2021; PMID: 34580178), we demonstrated that in the absence of ATAD2, there is reduced turnover of HIRAbound nucleosomes, as well as reduced nucleosome turnover, evidenced by the appearance of nucleosomes in regions that are normally nucleosome-free at active gene TSSs. We have no evidence supporting any other alternative hypothesis.

In the MS the reduced accessibility at active genes (Fig. 4) is attributed to H3.3 overloading. However, global changes in histone acetylation (e.g., H4K5ac) or other remodelers in KO cells could be also consider.

In fact, we meant that histone overloading could be responsible for the altered accessibility. This has been clearly demonstrated in case of S. cerevisiae in the absence of Yta7 (S.  cerevisiae’ ATAD2) (PMID: 25406467).

In relation with the sperm compaction assay (Fig. 8A), the DTT/heparin/Triton protocol may not fully reflect physiological decompaction. This could be validated with alternative methods (e.g., MNase sensitivity). 

The referee is right, but since this is a subtle effect as it can be judged by normal fertility, we doubt that milder approaches could reveal significant differences between wildtype and Atad2 KO sperms.

It is surprising that despite the observed alterations in the genome organization of the sperm, the natural fertility of the KO mice is not affected (Fig. 8C). This warrants deeper discussion: Is functional compensation occurring (e.g., by p97/VCP)? Analysis of epididymal sperm maturation or uterine environment could provide insights.

As detailed in the Discussion section, this work, together with our previous study (Wang et al., 2021; PMID: 34580178), highlights an overlooked level of regulation in histone chaperone activity: the release of chromatinbound factors following their interaction with chromatin. This is an energy-dependent process, driven by ATP and the associated ATPase activity of these factors. Such activity could be mediated by various proteins, such as p97/VCP or DNAJC9–HSP70, as discussed in the manuscript, or by yet unidentified factors. However, most of these mechanisms are likely to occur during the extensive histone-to-histone variant exchanges of meiosis and post-meiotic stages. To the best of our knowledge, epididymal sperm maturation and the uterine environment do not involve substantial histone-to-histone or histone-to-protamine exchanges.

The authors showed that MSCI genes present an enhancement of repression in the absence of ATAD2 by enhancing H3.3 function. It would be also of interest to analyze the behavior of the Sex body during its silencing (zygotene to pachytene) by looking at different markers (i.e., gamma-H2AX phosphorylation, Ubiquitylation etc). 

The referee is correct that this is an interesting question. Accordingly, in our future work, we plan to examine the sex body in more detail during its silencing, using a variety of relevant markers, including those suggested by the reviewer. However, we believe that such investigations fall outside the scope of the present study, which focuses on the molecular relationship between ATAD2 and H3.3, rather than on the role of H3.3 in regulating sex body transcription. For a comprehensive analysis of this aspect, studies should primarily focus on the H3.3 mouse models reported by Fontaine and colleagues (PMID: 35766398).

Fig. 6: Co-staining of TH2B/TP1/PRM1 is convincing but would benefit from quantification (% cells with overlapping signals).

The green cells correspond to those expressing TH2B but lacking transition proteins (TP) and protamine 1 (Prm1), indicating that they are at earlier stages than elongating–condensing spermatids. Counting these green cells simply reflects the ratio of elongating/condensing spermatids to earlier-stage cells, which varies depending on the field examined. The key point is that in wild-type mice, only red cells (elongating/condensing spermatids) and green cells (earlier stages) are observed. By contrast, in Atad2 KO testes, a significant proportion of yellow cells appears, which are never seen in wild-type tissue. The crucial metric is the percentage of yellow cells relative to the total number of elongating/condensing spermatids (red cells). In wild-type testes, this value is consistently 0%, whereas in Atad2 KO testes it always ranges between 50% and 100% across all fields containing substantial numbers of elongating/condensing spermatids.

relation s'organise entre anticipation psychique et confrontation perceptive

relation dominée par les attentes internes au détriment de l'altérité réelle

sujet impose ses anticipations au réel

Définition Hikikomori

Hikikomori = rupture entre virtule et perceptif

représentation anticipatrice

travail psychique implique acceptation du décalage entre attente et réalité

démarche scientifique = dynamique fonctionnement cognitif

adaptation psychique / circulation continue entre virtuel et perceptif

absence d'actualisation => présence relationnelle appauvrie ou inadéquate.

sans virtuel psychique , sujet submergé par immédiateté sensorielle.

notion actualisation désigne passage de la représentation à l'action concrète. Virtualisation permet la révision des représentations à partir de l'expériences vécue.

Lévy conceptualise la dynamique psychique = va et vient entre virtuel et action

relation humaine oscille entre ettentes internes et ajustement perceptif au réel

relation implique écart structurel entre anticipation et situation

perception ne fonctionne jamais seule : attentes du virtuel psychique

passage à l'imagination suppose prise de conscience et interruption volontaire du flux imaginaire

imagination est pensée prospective vers anticipation et projet

imagination articule représentation et action en vue d'une modification concrète du réel

objets internes issus expériences relationnelles précoces

travail psychique actif et structurant

rêvasseries s'inscrivent pas dans travail de symbolisation

mécanisme évitement psychique face à la souffrance

satisfaction illusoire

jeu constitue espace privilégié élaboration de l'imaginaire

imaginaire se distingue du virtuel par son absence de contrainte situationnelle

processus dynamique

Le virtuel psychique doit maintenir un écart ajusté au réel pour rester fonctionnel

virtuel psychique composé anticipations qui orientent l'action anvant la rencontre avec le réel

tripartition des représentations

distinction essentielle

technologies numériques matérialisent des processus psychiques préexistants

immédiateté de l'actualisation permet de comprendre l'attrait du numérique

virtuel s'oppose à l'actuel non au réel

enjeu : continuité

stabilité relative de l'objet => construction de représentations internes

virtuel psychique n'existe pas en l'absence de l'objet

conception deleuzienne du virtuel

Distingue le virtuel du potentiel

Virtuel = potentiel. critiqué par Deleuze.

Rappel conceptuel : virtuel précède numérique. forme d'actualisation.

Distinction centrale : virtuel renvoie une dimension psychique réelle et non imaginaire. Assimilation du numérique à irréel.

Spécificité interactive du numérique => fort pouvoir d'engagement cognitif et émotionnel. Eclaire les mécanismes décrits par young.

Complexité des rapports virtuels numérique => idée usages numériques ne peuvent être évalués indépendamment du contexte psychique et social.

Penser le numérique comme une transformation des modalités de présence sociale.

modalité de présence remet en cause l’opposition classique virtuel/réel. 1 interaction est réelle dès lors qu’elle engage des affects. Echanges numériques mobilisent les mêmes processus sociaux fondamentaux que les interactions en présence.

Concept de virtuel ne renvoie pas au support mais à une construction psychique. Clarification conceptuelle essentielle pour éviter une pathologisation abusive des usages numériques. Virtuel désigne un espace de représentation et de symbolisation, non un artefact technologique.

Rvo

désarticulation sociale

désarticulation psychique

perceptif

imaginaire

virtuel psychique

ethymologie du mot virtuel

Construction psychique du virtuel

Références de l'ouvrage

eLife Assessment

This useful study reports a method to detect and analyze a novel post-translational modification, lysine acetoacetylation (Kacac), finding it regulates protein metabolism pathways. The study unveils epigenetic modifiers involved in placing this mark, including key histone acetyltransferases such as p300, and concomitant HDACs, which remove the mark. Proteomic and bioinformatics analysis identified many human proteins with Kacac sites, potentially suggesting broad effects on cellular processes and disease mechanisms. The data presented are solid and the study will be of interest to those studying protein and metabolic regulation.

Reviewer #3 (Public review):

Summary:

This paper presents a timely and significant contribution to the study of lysine acetoacetylation (Kacac). The authors successfully demonstrate a novel and practical chemo-immunological method using the reducing reagent NaBH4 to transform Kacac into lysine β-hydroxybutyrylation (Kbhb).

Strengths:

This innovative approach enables simultaneous investigation of Kacac and Kbhb, showcasing their potential in advancing our understanding of post-translational modifications and their roles in cellular metabolism and disease.

Weaknesses:

The study lacks supporting in vivo data, such as gene knockdown experiments, to validate the proposed conclusions at the cellular level.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #2 (Public review):

In the manuscript by Fu et al., the authors developed a chemo-immunological method for the reliable detection of Kacac, a novel post-translational modification, and demonstrated that acetoacetate and AACS serve as key regulators of cellular Kacac levels. Furthermore, the authors identified the enzymatic addition of the Kacac mark by acyltransferases GCN5, p300, and PCAF, as well as its removal by deacetylase HDAC3. These findings indicate that AACS utilizes acetoacetate to generate acetoacetyl-CoA in the cytosol, which is subsequently transferred into the nucleus for histone Kacac modification. A comprehensive proteomic analysis has identified 139 Kacac sites on 85 human proteins. Bioinformatics analysis of Kacac substrates and RNA-seq data reveal the broad impacts of Kacac on diverse cellular processes and various pathophysiological conditions. This study provides valuable additional insights into the investigation of Kacac and would serve as a helpful resource for future physiological or pathological research.

The authors have made efforts to revise this manuscript and address my concerns. The revisions are appropriate and have improved the quality of the manuscript.

We appreciate the constructive and thoughtful feedbacks, which have been invaluable in enhancing the quality of our manuscript.

Reviewer #3 (Public review):

Summary:

This paper presents a timely and significant contribution to the study of lysine acetoacetylation (Kacac). The authors successfully demonstrate a novel and practical chemoimmunological method using the reducing reagent NaBH4 to transform Kacac into lysine βhydroxybutyrylation (Kbhb).

Thank you for the positive and insightful comments.

Strengths:

This innovative approach enables simultaneous investigation of Kacac and Kbhb, showcasing its potential in advancing our understanding of post-translational modifications and their roles in cellular metabolism and disease.

We are grateful for the reviewer’s comments, which has contributed to enhancing the quality of our study.

Weaknesses:

The experimental evidence presented in the article is insufficient to fully support the authors' conclusions. In the in vitro assays, the proteins used appear to be highly inconsistent with their expected molecular weights, as shown by Coomassie Brilliant Blue staining (Figure S3A). For example, p300, which has a theoretical molecular weight of approximately 270 kDa, appeared at around 37 kDa; GCN5/PCAF, expected to be ~70 kDa, appeared below 20 kDa. Other proteins used in the in vitro experiments also exhibited similarly large discrepancies from their predicted sizes. These inconsistencies severely compromise the reliability of the in vitro findings. Furthermore, the study lacks supporting in vivo data, such as gene knockdown experiments, to validate the proposed conclusions at the cellular level.

We appreciate the reviewer’s comments. In the biochemical assays, we used the expressed catalytic domains of HATs—rather than the full-length proteins for activity testing. Specifically, the following constructs were expressed and purified: p300 (1287– 1666), GCN5 (499-663), PCAF (493-658), MOF (125-458), MOZ (497-780), MBP-MORF (361-716), Tip60 (221-512), HAT1 (20-341), and HBO1 (full length). This resulted in the observed discrepancies in molecular weight in Figure S3A compared to the expected fulllength weights. 

Although a recent study (PMID: 37382194) reported the acetoacetyltransferase activities of p300 and GCN5 in cells, we recognize that additional knockdown experiments would be necessary to substantiate their contributions to in vivo Kacac generation and to explore the functional roles of Kacac in an enzyme-specific context. We plan to address these kinds of research issues in our future work.

Besluit dat data.overheid.nl aanwijst als centraal informatiepunt ihkv de DGA

Advies AP mbt Uitvoeringsbesluit datagovernanceverordening. Merkt op dat CBS bij het inzetten van de beveiligde verwerkingsomgeving een verwerker wordt, en een verwerkingsovereenkomst nodig zal zijn.

RvS heeft geen opmerkingen bij Uitvoeringsbesluit Datagovernanceverordening

Uitvoeringsbesluit Datagovernanceverordening Regelt bevoegde organen, en enkele elementen rondom vergoedingen. RvS en AP hebben advies uitgebracht.

１

１

A Caesar cipher, also known as Caesar's cipher, the shift cipher, Caesar's code, or the Caesar shift, is one of the simplest and most widely known encryption techniques in

Python?

Of course in Python we can add instance variables to a class at any time by simply assigning a value to objectReferenc.variableName

légitime scientifiquement la notion d'addiction non chimique

Notion de retentissement durable constitue un critère clinique : altération des fonctions exécutives (inhibition, planification) et priorisation excessive des stimulations numériques des stimulations numériques dans le système attentionnel. Permet de distinguer un usage intensif d'un usage pathologique

Definition de l'addiction : rapproche les usages numériques des addictions comportementales. Rejoint la conceptualisation de Young : addiction à Internet repose moins sur l’objet que sur l’altération des processus de régulation cognitive et motivationnelle. Prudence terminologique adoptée par Mouren anticipe la sur-pathologisation des usages numériques chez les jeunes.

Distinction enfant /adulte

retentissement fonctionnel

prudence terminologique

Hier wordt de userstory gegeven voor het importeren van de nieuwe situatie neem ik aan. In het beschreven proces zie ik ook de behoefte om het projectgebied van de huidige situatie te exporteren naar een NLCS tekening. Bij de beschrijvingen zou het duidelijker zijn als toegevoegd wordt wanneer je spreekt over huidige situatie en wanneer over de nieuwe of aangepaste situatie van de riolering.

volgens mij mag de userstory nog geen oplossingen beschrijven (toch?)

Mogelijke toevoeging: bij elke beslissing die de belanghebbenden nemen willen ze kunnen vertrouwen op de assetadministratie, dat veronderstelt dat de werkelijkheid 1 op 1 weergegeven is in de administratie.

Ik lees in deze paragraaf weinig over wensen en verwachtingen. Ik kan mij voorstellen dat iedere deelnemer in het revisieproces de wens heeft dat wat in werkelijkheid gebouwd wordt ook 1 op 1 overgenomen wordt in de Asset administratie.

DOI: 10.7554/eLife.107103

Resource: GraphPad Prism (RRID:SCR_002798)

Curator: @evieth

SciCrunch record: RRID:SCR_002798

What is this?

DOI: 10.7554/eLife.107103

Resource: R package: maxstat (RRID:SCR_025679)

Curator: @scibot

SciCrunch record: RRID:SCR_025679

What is this?

DOI: 10.3390/vaccines13111103

Resource: (NIH Nonhuman Primate Reagent Resource Cat# PR-0407, RRID:AB_2716322)

Curator: @giovanni.decastro

SciCrunch record: RRID:AB_2716322

What is this?

DOI: 10.3390/vaccines13111103

Resource: (NIH Nonhuman Primate Reagent Resource Cat# PR-7114, RRID:AB_2819312)

Curator: @giovanni.decastro

SciCrunch record: RRID:AB_2819312

What is this?

DOI: 10.3390/vaccines13111103

Resource: None

Curator: @giovanni.decastro

SciCrunch record: RRID:AB_2895609

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 9272, RRID:AB_329827)

Curator: @evieth

SciCrunch record: RRID:AB_329827

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 12790, RRID:AB_2631166)

Curator: @evieth

SciCrunch record: RRID:AB_2631166

What is this?

DOI: 10.1177/15347354251401187

Resource: None

Curator: @evieth

SciCrunch record: RRID:AB_2799796

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 3716, RRID:AB_2116962)

Curator: @evieth

SciCrunch record: RRID:AB_2116962

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 15108, RRID:AB_2798711)

Curator: @scibot

SciCrunch record: RRID:AB_2798711

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 9662, RRID:AB_331439)

Curator: @scibot

SciCrunch record: RRID:AB_331439

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 9502, RRID:AB_2068621)

Curator: @scibot

SciCrunch record: RRID:AB_2068621

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 3852, RRID:AB_2144868)

Curator: @scibot

SciCrunch record: RRID:AB_2144868

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 2546, RRID:AB_2276129)

Curator: @scibot

SciCrunch record: RRID:AB_2276129

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 4706, RRID:AB_823544)

Curator: @scibot

SciCrunch record: RRID:AB_823544

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 2032, RRID:AB_2136278)

Curator: @scibot

SciCrunch record: RRID:AB_2136278

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 67955, RRID:AB_2909603)

Curator: @scibot

SciCrunch record: RRID:AB_2909603

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 9102, RRID:AB_330744)

Curator: @scibot

SciCrunch record: RRID:AB_330744

What is this?

DOI: 10.1177/15347354251401187

Resource: (Enzo Life Sciences Cat# ADI-905-733, RRID:AB_10616090)

Curator: @scibot

SciCrunch record: RRID:AB_10616090

What is this?

DOI: 10.1177/15347354251401187

Resource: GraphPad Prism (RRID:SCR_002798)

Curator: @scibot

SciCrunch record: RRID:SCR_002798

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 2118, RRID:AB_561053)

Curator: @scibot

SciCrunch record: RRID:AB_561053

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 7237, RRID:AB_10895832)

Curator: @scibot

SciCrunch record: RRID:AB_10895832

What is this?

DOI: 10.1177/15347354251401187

Resource: (Cell Signaling Technology Cat# 9496, RRID:AB_561381)

Curator: @scibot

SciCrunch record: RRID:AB_561381

What is this?

DOI: 10.1177/15347354251401187

Resource: NovoExpress (RRID:SCR_024676)

Curator: @scibot

SciCrunch record: RRID:SCR_024676

What is this?

DOI: 10.1177/15347354251401187

Resource: (ATCC Cat# TIB-71, RRID:CVCL_0493)

Curator: @scibot

SciCrunch record: RRID:CVCL_0493

What is this?

DOI: 10.1158/2326-6066.CIR-25-0062

Resource: Squidpy (RRID:SCR_026157)

Curator: @evieth

SciCrunch record: RRID:SCR_026157

What is this?

DOI: 10.1155/humu

Resource: (IZSLER Cat# BS TCL 4, RRID:CVCL_0186)

Curator: @dhovakimyan1

SciCrunch record: RRID:CVCL_0186

What is this?

DOI: 10.1111/imcb.70061

Resource: Nonhuman Primate Reagent Resource (RRID:SCR_012986)

Curator: @giovanni.decastro

SciCrunch record: RRID:SCR_012986

What is this?

DOI: 10.1093/jimmun/vkaf328

Resource: None

Curator: @dhovakimyan1

SciCrunch record: RRID:Addgene_21050

What is this?

DOI: 10.1093/jimmun/vkaf328

Resource: RRID:Addgene_10841

Curator: @dhovakimyan1

SciCrunch record: RRID:Addgene_10841

What is this?

DOI: 10.1093/jimmun/vkaf328

Resource: (IMSR Cat# JAX_002216,RRID:IMSR_JAX:002216)

Curator: @dhovakimyan1

SciCrunch record: RRID:IMSR_JAX:002216

What is this?

DOI: 10.1038/s44318-025-00634-7

Resource: Adobe Photoshop (RRID:SCR_014199)

Curator: @evieth

SciCrunch record: RRID:SCR_014199

What is this?

DOI: 10.1016/j.jbc.2025.111074

Resource: RRID:IMSR_JAX:000664

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

DOI: 10.1016/j.isci.2025.114444

Resource: Python Programming Language (RRID:SCR_008394)

Curator: @areedewitt04

SciCrunch record: RRID:SCR_008394

What is this?

DOI: 10.1016/j.isci.2025.114305

Resource: RRID:Addgene_21179

Curator: @areedewitt04

SciCrunch record: RRID:Addgene_21179

What is this?

DOI: 10.1016/j.isci.2025.114305

Resource: RRID:Addgene_12260

Curator: @areedewitt04

SciCrunch record: RRID:Addgene_12260

What is this?

DOI: 10.1016/j.isci.2025.114305

Resource: (ATCC Cat# CRL-2302, RRID:CVCL_0145)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_0145

What is this?

DOI: 10.1016/j.isci.2025.114284

Resource: (IMSR Cat# JAX_001303,RRID:IMSR_JAX:001303)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:001303

What is this?

DOI: 10.1016/j.isci.2025.114284

Resource: (ATCC Cat# CRL-10317, RRID:CVCL_0598)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_0598

What is this?

DOI: 10.1016/j.cmet.2025.11.011

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_GPT:T025446

What is this?

DOI: 10.1016/j.cmet.2025.11.011

Resource: RRID:Addgene_98895

Curator: @areedewitt04

SciCrunch record: RRID:Addgene_98895

What is this?

DOI: 10.1016/j.cmet.2025.11.011

Resource: RRID:Addgene_26475

Curator: @areedewitt04

SciCrunch record: RRID:Addgene_26475

What is this?

DOI: 10.1016/j.cmet.2025.11.011

Resource: (RRID:CVCL_0063)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_0063

What is this?

DOI: 10.1016/j.cmet.2025.11.011

Resource: (CLS Cat# 300342/p657_SK-OV-3, RRID:CVCL_0532)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_0532

What is this?

DOI: 10.1016/j.chom.2025.11.015

Resource: (RCB Cat# RCB0988, RRID:CVCL_0025)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_0025

What is this?

DOI: 10.1016/j.chom.2025.11.015

Resource: (RRID:CVCL_1926)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_1926

What is this?

DOI: 10.1016/j.chom.2025.11.015

Resource: (IMSR Cat# GPT_N000013,RRID:IMSR_GPT:N000013)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_GPT:N000013

What is this?

DOI: 10.1016/j.celrep.2025.116745

Resource: (RRID:CVCL_0006)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_0006

What is this?

DOI: 10.1016/j.celrep.2025.116745

Resource: (BCRC Cat# 60005, RRID:CVCL_0030)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_0030

What is this?

DOI: 10.1016/j.celrep.2025.116745

Resource: (ATCC Cat# PTA-6671, RRID:CVCL_3755)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_3755

What is this?

DOI: 10.1016/j.celrep.2025.116745

Resource: (ATCC Cat# CRL-10317, RRID:CVCL_0598)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_0598

What is this?

DOI: 10.1016/j.celrep.2025.116745

Resource: (RRID:CVCL_1926)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_1926

What is this?

DOI: 10.1016/j.celrep.2025.116745

Resource: RRID:Addgene_12259

Curator: @areedewitt04

SciCrunch record: RRID:Addgene_12259

What is this?

DOI: 10.1016/j.celrep.2025.116719

Resource: (IMSR Cat# JAX_017525,RRID:IMSR_JAX:017525)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:017525

What is this?

DOI: 10.1016/j.celrep.2025.116719

Resource: RRID:IMSR_JAX:007909

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:007909

What is this?

DOI: 10.1016/j.celrep.2025.116703

Resource: (IMSR Cat# JAX_012606,RRID:IMSR_JAX:012606)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:012606

What is this?

DOI: 10.1016/j.celrep.2025.116703

Resource: (IMSR Cat# CRL_207,RRID:IMSR_CRL:207)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_CRL:207

What is this?

DOI: 10.1016/j.celrep.2025.116703

Resource: (IMSR Cat# JAX_005104,RRID:IMSR_JAX:005104)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:005104

What is this?

DOI: 10.1016/j.cell.2025.11.022

Resource: (KCLB Cat# 80008, RRID:CVCL_0159)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_0159

What is this?

DOI: 10.1007/s12035-025-05433-z

Resource: (KCB Cat# KCB 92029YJ, RRID:CVCL_0464)

Curator: @dhovakimyan1

SciCrunch record: RRID:CVCL_0464

What is this?

DOI: 10.1002/ctm2.70539

Resource: RSeQC (RRID:SCR_005275)

Curator: @evieth

SciCrunch record: RRID:SCR_005275

What is this?

DOI: 10.1002/advs.202519735

Resource: (RRID:CVCL_B288)

Curator: @dhovakimyan1

SciCrunch record: RRID:CVCL_B288

What is this?

DOI: 10.1002/advs.202519735

Resource: (KCB Cat# KCB 200848YJ, RRID:CVCL_0546)

Curator: @dhovakimyan1

SciCrunch record: RRID:CVCL_0546

What is this?

DOI: 10.1002/advs.202519735

Resource: (CLS Cat# 300266/p487_LOVO, RRID:CVCL_0399)

Curator: @dhovakimyan1

SciCrunch record: RRID:CVCL_0399

What is this?

DOI: 10.1002/advs.202512278

Resource: RRID:IMSR_GPT:T003822

Curator: @dhovakimyan1

SciCrunch record: RRID:IMSR_GPT:T003822

What is this?

DOI: 10.7554/eLife.90531

Resource: (IMSR Cat# JAX_006584,RRID:IMSR_JAX:006584)

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:006584

What is this?

DOI: 10.7554/eLife.90531

Resource: RRID:IMSR_JAX:000664

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

DOI: 10.7554/eLife.90531

Resource: (IMSR Cat# JAX_002288,RRID:IMSR_JAX:002288)

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:002288

What is this?

DOI: 10.7554/eLife.90531

Resource: (IMSR Cat# JAX_000651,RRID:IMSR_JAX:000651)

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:000651

What is this?

DOI: 10.7554/eLife.105271

Resource: Inkscape (RRID:SCR_014479)

Curator: @scibot

SciCrunch record: RRID:SCR_014479

What is this?

DOI: 10.7554/eLife.105271

Resource: None

Curator: @scibot

SciCrunch record: RRID:SCR_027660

What is this?

DOI: 10.7554/eLife.105271

Resource: GraphPad Prism (RRID:SCR_002798)

Curator: @scibot

SciCrunch record: RRID:SCR_002798

What is this?

DOI: 10.7554/eLife.105271

Resource: Imaris (RRID:SCR_007370)

Curator: @scibot

SciCrunch record: RRID:SCR_007370

What is this?

DOI: 10.7554/eLife.105271

Resource: ImageJ (RRID:SCR_003070)

Curator: @scibot

SciCrunch record: RRID:SCR_003070

What is this?

DOI: 10.7554/eLife.105271

Resource: Seurat (RRID:SCR_016341)

Curator: @scibot

SciCrunch record: RRID:SCR_016341

What is this?

DOI: 10.7554/eLife.105271

Resource: Patchmaster (RRID:SCR_000034)

Curator: @scibot

SciCrunch record: RRID:SCR_000034

What is this?

DOI: 10.7554/eLife.105271

Resource: ZEISS ZEN Microscopy Software (RRID:SCR_013672)

Curator: @scibot

SciCrunch record: RRID:SCR_013672

What is this?

DOI: 10.7554/eLife.105271

Resource: (Thermo Fisher Scientific Cat# A-11010, RRID:AB_2534077)

Curator: @scibot

SciCrunch record: RRID:AB_2534077

What is this?

DOI: 10.7554/eLife.105271

Resource: (Thermo Fisher Scientific Cat# A-11073, RRID:AB_2534117)

Curator: @scibot

SciCrunch record: RRID:AB_2534117

What is this?

DOI: 10.7554/eLife.105271

Resource: (Thermo Fisher Scientific Cat# A-11008, RRID:AB_143165)

Curator: @scibot

SciCrunch record: RRID:AB_143165

What is this?

DOI: 10.7554/eLife.105271

Resource: (Thermo Fisher Scientific Cat# A-21235, RRID:AB_2535804)

Curator: @scibot

SciCrunch record: RRID:AB_2535804

What is this?

DOI: 10.7554/eLife.105271

Resource: (Thermo Fisher Scientific Cat# A-11007, RRID:AB_10561522)

Curator: @scibot

SciCrunch record: RRID:AB_10561522

What is this?

DOI: 10.7554/eLife.105271

Resource: (Thermo Fisher Scientific Cat# A-11017, RRID:AB_2534084)

Curator: @scibot

SciCrunch record: RRID:AB_2534084

What is this?

DOI: 10.7554/eLife.105271

Resource: (Thermo Fisher Scientific Cat# A-11039, RRID:AB_2534096)

Curator: @scibot

SciCrunch record: RRID:AB_2534096

What is this?

DOI: 10.7554/eLife.105271

Resource: (Synaptic Systems Cat# 101 004, RRID:AB_1210382)

Curator: @scibot

SciCrunch record: RRID:AB_1210382

What is this?

DOI: 10.7554/eLife.105271

Resource: (Synaptic Systems Cat# 131 002, RRID:AB_887871)

Curator: @scibot

SciCrunch record: RRID:AB_887871

What is this?

DOI: 10.7554/eLife.105271

Resource: (Millipore Cat# MAB386, RRID:AB_94975)

Curator: @scibot

SciCrunch record: RRID:AB_94975

What is this?

DOI: 10.7554/eLife.105271

Resource: (Synaptic Systems Cat# 377 005, RRID:AB_2721101)

Curator: @scibot

SciCrunch record: RRID:AB_2721101

What is this?

DOI: 10.7554/eLife.105271

Resource: (Thermo Fisher Scientific Cat# PA5-78397, RRID:AB_2736178)

Curator: @scibot

SciCrunch record: RRID:AB_2736178

What is this?

DOI: 10.7554/eLife.105271

Resource: (Thermo Fisher Scientific Cat# M11217, RRID:AB_2536611)

Curator: @scibot

SciCrunch record: RRID:AB_2536611

What is this?

DOI: 10.7554/eLife.105271

Resource: (Abcam Cat# ab13970, RRID:AB_300798)

Curator: @scibot

SciCrunch record: RRID:AB_300798

What is this?

DOI: 10.7554/eLife.105271

Resource: RRID:IMSR_JAX:007909

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:007909

What is this?

DOI: 10.7554/eLife.105271

Resource: (IMSR Cat# JAX_016962,RRID:IMSR_JAX:016962)

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:016962

What is this?

DOI: 10.7554/eLife.105271

Resource: (Millipore Cat# MAB318, RRID:AB_2201528)

Curator: @scibot

SciCrunch record: RRID:AB_2201528

What is this?

DOI: 10.7554/eLife.105271

Resource: RRID:MGI:3028467

Curator: @scibot

SciCrunch record: RRID:MGI:3028467

What is this?

DOI: 10.7554/eLife.105271

Resource: RRID:IMSR_JAX:006660

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:006660

What is this?

DOI: 10.7554/eLife.103705

Resource: RRID:BDSC_3605

Curator: @scibot

SciCrunch record: RRID:BDSC_3605

What is this?

DOI: 10.7554/eLife.103705

Resource: FlowJo (RRID:SCR_008520)

Curator: @scibot

SciCrunch record: RRID:SCR_008520

What is this?

DOI: 10.7554/eLife.103705

Resource: GraphPad Prism (RRID:SCR_002798)

Curator: @scibot

SciCrunch record: RRID:SCR_002798

What is this?

DOI: 10.7554/eLife.103705

Resource: Fiji (RRID:SCR_002285)

Curator: @scibot

SciCrunch record: RRID:SCR_002285

What is this?

DOI: 10.3389/fimmu.2025.1671961

Resource: (Proteintech Cat# SA00001-2, RRID:AB_2722564)

Curator: @scibot

SciCrunch record: RRID:AB_2722564

What is this?

DOI: 10.3389/fimmu.2025.1671961

Resource: (Proteintech Cat# SA00001-1, RRID:AB_2722565)

Curator: @scibot

SciCrunch record: RRID:AB_2722565

What is this?

DOI: 10.3389/fimmu.2025.1671961

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2138307

What is this?

DOI: 10.3389/fimmu.2025.1671961

Resource: (Proteintech Cat# 60268-1-Ig, RRID:AB_2881388)

Curator: @scibot

SciCrunch record: RRID:AB_2881388

What is this?

DOI: 10.3389/fimmu.2025.1671961

Resource: (Proteintech Cat# 20145-1-AP, RRID:AB_10699877)

Curator: @scibot

SciCrunch record: RRID:AB_10699877

What is this?

DOI: 10.3389/fimmu.2025.1671961

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2884207

What is this?

DOI: 10.26508/lsa.202503508

Resource: Northwestern University Feinberg School of Medicine Center for Advanced Microscopy Nikon Imaging Center Core Facility (RRID:SCR_020996)

Curator: @scibot

SciCrunch record: RRID:SCR_020996

What is this?

DOI: 10.2478/raon-2025-0057

Resource: None

Curator: @scibot

SciCrunch record: RRID:CVCL_0D71

What is this?

DOI: 10.2478/raon-2025-0057

Resource: (Millipore Cat# SCC073, RRID:CVCL_7773)

Curator: @scibot

SciCrunch record: RRID:CVCL_7773

What is this?

DOI: 10.2478/raon-2025-0057

Resource: (BCRJ Cat# 0301, RRID:CVCL_1218)

Curator: @scibot

SciCrunch record: RRID:CVCL_1218

What is this?

DOI: 10.2478/raon-2025-0057

Resource: (DSMZ Cat# ACC-670, RRID:CVCL_1899)

Curator: @scibot

SciCrunch record: RRID:CVCL_1899

What is this?

DOI: 10.1371/journal.pone.0338884

Resource: IBM SPSS Statistics (RRID:SCR_016479)

Curator: @scibot

SciCrunch record: RRID:SCR_016479

What is this?

DOI: 10.1371/journal.pone.0338623

Resource: Applied Biosystems 3730 Genetic Analyzer (RRID:SCR_018052)

Curator: @scibot

SciCrunch record: RRID:SCR_018052

What is this?

DOI: 10.1371/journal.pbio.3003553

Resource: RRID:BDSC_55

Curator: @scibot

SciCrunch record: RRID:BDSC_55

What is this?

DOI: 10.1371/journal.pbio.3003553

Resource: RRID:BDSC_602

Curator: @scibot

SciCrunch record: RRID:BDSC_602

What is this?

DOI: 10.1371/journal.pbio.3003553

Resource: RRID:BDSC_65

Curator: @scibot

SciCrunch record: RRID:BDSC_65

What is this?