Animal cell culture methods

Spheroplast resuspension buffer0.1M KCl15 mM HEPES (pH 7.5)3 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid(EGTA)10% GlycerolPhosphatidylinositol sonication buffer10 mM HEPES (pH 7.5)1 mM EGTA PI3-kinase reaction buffer40 mM HEPES (pH 7.5)20 mM MgCl280 μM ATP5 μCi γ-P32ATPDeveloping solution for thin layer chromatography(120.2 ml)Chloroform –60 mlMethanol –47 mlAmmonia –4.4 mlWater –8.8 ml

Reagents for PI3-kinase assay

SDS-loading buffer was prepared as a 4X stock solutionin H2Oand used at a 1X concentration.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSRunning buffer was preparedas a 10X stock solution and diluted to 1X concentration before use.Buffers for Western blotanalysisTransfer buffer (10X stock solution)0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSTransfer buffer was prepared as a 10X stock solution and diluted to 1X concentration.1X Transfer buffer (1 litre)200 ml of methanol100 ml of 10X transfer buffer700 ml of waterTris-BufferSaline (TBS)25 mM Tris150 mM NaClpH was adjusted to 7.4 with HCl.TBS buffer was prepared asa10X stock solution and diluted to 1X concentration.Blocking and wash buffers (PBS-T and TBS-T)5% Fat-free milk0.1% Tween-20Volume was made to 100 ml with 1X TBS

1 mM sodium orthovanadate1 X protease inhibitor cocktail SDS-PAGE30% Acrylamide solution29 g Acrylamide1 gBis-acrylamideDissolved in 100 ml H2O.10% Sodium Dodecyl Sulfate (SDS)10 g SDS in 100 ml H2OResolving gel mix (12%) (20 ml)6.6 ml H2O8 ml 30% acrylamide:bisacrylamide (29:1) mix5 ml 1.5 M Tris-HCl (pH 8.8)200 μl 10% SDS200 μl 10% Ammonium persulfate(APS)8 μl N,N,N′,N′-Tetramethylethylenediamine(TEMED)Stacking gel mix (5%, 6 ml)4.1 ml H2O1 ml 30% acrylamide:bisacrylamide (29:1) mix750 μl 1 M Tris-HCl (pH 6.8)60 μl 10% SDS60 μl 10% APS6 μl TEMEDSDS loading buffer130 mM Tris-HCl (pH 8.0)20% (v/v) Glycerol4.6% (w/v) SDS0.02% Bromophenol Blue2% DTT

Whole cell lysis buffer (Homogenizing buffer)50 mM Tris-HCl (pH 7.5)2 mM EDTA10 mM sodium fluoride

Buffers for protein extraction and analysis by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

150 mM NaCl1% Triton-X1% SDSBuffer B50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 MSorbitol50 mM β-mercaptoethanol (To be added just before use)Buffer C100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSAE buffer3 M Sodium acetate(pH 5.3)0.5 M EDTA (pH 8.0)Phenol:Chloroform:Isoamyl alcohol (25:24:1) solution25 ml Tris-equilibrated Phenol24 ml Chloroform1 ml Isoamyl alcholDNA sample loading buffer0.25% Bromophenol blue0.25% Xylene cyanol15% FicollDNA sample loading buffer was prepared in water

Buffer A50 mM Tris-HCl(pH 8)10 mM EDTA

Buffers for extraction and analysis of genomic DNA and RNA

Stripping solutionfor DNA1% SDS0.1% SSCDesired volume was adjusted with sterile water. Alternatively, 0.4 M NaOH was also used to stripthe bound probes fromnylon membranes.HEPES [4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid] buffer1 M HEPESpH was adjusted to 7.5 with NaOH.HEPES was used as a buffering agent for preparing plates of YNB medium of different pH. Buffer was filter-sterilized and stored in an amber-coloured bottle.INOUE transformation buffer10 mM PIPES15 mM CaCl2.2H2O250 mM KCl55 mM MnCl2.4H2OpH was adjusted to 6.7 with 1 N KOH.Yeast transformation reagents1 M Lithium acetate 50% Polyethylene glycol2 mg/ml carrier DNADimethyl sulfoxide (DMSO)Zymolyase cocktail buffer for yeast colony PCR2.5 mg/ml Zymolyase1.2 M SorbitolZymolyase buffer was prepared in 1X PBS

pH was adjusted to 8.5 with glacial acetic acid.TAE buffer was prepared asa50Xstock solution and used at 0.5X concentration.Alkaline denaturing solution for DNAfor membrane preparation0.5 M NaCl0.25 M NaOHVolume was adjusted with sterile water.Denhardt’s solution (50X)1%Ficoll-4001% Polyvinyl pyrollidone1% Bovine serum albuminVolume was adjusted with water and solution was stored at -20°C.Saline Sodium Citrate (SSC) buffer(20X)3.0 M Sodium chloride0.3 M Sodium citrate Volume was adjusted with water and solution was sterilized by autoclaving.Prehybridization Buffer5X SSC5X Denhardt’s solution50% Filtered formamide1% SDSVolume was adjusted with sterile water.Post hybridization wash buffersWash buffer 12X SSC0.1% SDSWash buffer21X SSC0.1% SDS

Phosphate-Buffered Saline (PBS)137 mM NaCl2.7 mM KCl10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3 before autoclaving.PBS was prepared as a 10X stock solution and diluted to 1X concentration before autoclaving.Tris-HCl buffer0.5 M Trizma BasepH was adjusted to 7.6 using concentrated HCl.Tris-Cl buffer was prepared as a 10Xstock solution and used at a 1X concentration.Tris-EDTA (TE) buffer10 mM Tris-HCl (pH 8.0)1 mM EDTATris-Acetic acid EDTA (TAE) buffer40 mM Tris base0.5 M EDTA

Common buffers

Buffers and solutions

10 mM NaCl2.5 mM KCl10 mM MgCl210 mM MgSO4LB-ampicillin and LB-kanamycin platesLBmedium50 μg/ml ampicillin30 μg/ml kanamycinMedia and solutions were sterilizedeither by routine autoclaving at 121°C and 15 psi for 20 minor by filtration through membrane of 0.22 μm porosity

Luria Bertani (LB)0.5% Yeast Extract1% Tryptone1% NaClSuper Optimal Broth (SOB)0.5% Yeast Extract2% Peptone

Bacterial media

Yeast extract Peptone Dextrose (YPD)1% Yeast extract2% Peptone2% DextroseYeast Nitrogen Base (YNB)0.67% Yeast Nitrogen Base2% DextroseFor alternate carbon source utilization experiments, dextrose was replaced withother carbon sourcesviz.,sodium acetate, ethanol, oleic acid, glycerol and citric acid.Yeast Nitrogen Base (YNB) without ammonium sulphate and amino acids0.17% Yeast Nitrogen Base2% DextroseCasamino Acid (CAA)0.67% Yeast Nitrogen Base2% Dextrose0.6% Casamino acidsFor preparing plates, 2% agar was added tothe medium before autoclaving

Yeast media

Media

Table 2.4: List of the oligonucleotides used to confirm deletion of C. glabrataORFs

Table 2.3: List of the oligonucleotides used in the study

Table2.2: List of the antibodies used in the study

Table 2.1: List of strains and plasmidsused in the study

methanol, acetic acid, potassium dihydrogen orthrophosphate, dipotassium hydrogen phosphate, disodium hydrogen orthrophosphate, acetone and citric acid were purchased from Qualigen chemicals. Fluconazole was procured from Ranbaxy.Lysotracker-Red DND 99 and FM 4-64 were obtained from Molecular Probes. Hybond-N and Hybond-P membranes for nucleic acid and protein transfer, respectively, were purchased from Amersham Biosciences. SYBR-green kit for real-time PCR was procured from Eurogentech. Superscipt SS-III RT kit and Pfu polymerase were obtained from Invitrogen. Different restriction enzymes used for cloning and knock-out generation were purchased from New England Biolabs (NEB). High fidelity DNA Pfx polymerase waspurchased fromFinnzymes. Plasmid DNA purification, PCR purification, gel extraction and reaction clean up kits were procured from Qiagen.Medium components for C. glabrataand bacterial culture viz.,yeast extract, peptone, tryptone, cassamino acid hydrolysate, yeast nitrogen base, yeast nitrogen base without ammonium sulphate, yeast nitrogen base without ammonium sulphate and amino acids and yeast carbon basewere purchased from BD (Becton, Dickinson and Company, USA). Animal cell culture media RPMI-1640, DMEM and α-MEM were procured from Hyclone. Fetal bovine serum, glutamine and antibiotics for cell culture medium were obtained from Gibco-Invitrogen

Agarose, phenol, dimethyl sulphoxide (DMSO), sodium acetate, sodium chloride, sodium hydroxide, sodium carbonate, sodium bicarbonate, trizma base, sodium dodecyl sulphate (SDS), formamide, calcium chloride, ethylenediaminetetraacetic acid(EDTA), glycerol, polyethylene glycol, ficoll, diphenyleneiodinium (DPI), methyl methanesulphonate (MMS), camptothecin, hydroxyurea, ammonium persulphate, TEMED, acrylamide, bis-acrylamide, coomassie brilliant blue (CBB), chloroform, formaldehyde, glycine, lithium chloride, lithium acetate, menadione, isopropanol, phorbol myrsityl acetate (PMA), nuclease free water, wortmannin, bafilomycin-A, diethylpyrocarbonate (DEPC), orthrophenylenediamine (OPD), tween-20, acid washed glass beads, trypan blue, Taq DNA Polymease, trisodium citrate dihydrate and uracil were purchased from Sigma Chemicals. β-mercaptoethanol was obtained from GE Biosciences.Protease inhibitor tablets were procured from Roche. Dextrose, sucrose, agar, ammonium sulphate, potassium chloride, caffeine, magnesium chloride and sorbitol were obtained from Himedia.Hydrogen peroxide, hydrochloric acid, sulphuric acid,

Chemicals, kits and culture medium components

Oligonucleotides used in this study were designed either by freely available online tool Primer 3 plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/)or Generunner software. Oligonucleotides were commercially synthesised at MWG Biotech Pvt. Ltd., Bangalore, India. Oligonucleotides used in this study are listed in Table 2.3

Oligonucleotides

All antibodies, their sources, clonality and dilutions used are listed in Table 2.2

Antibodies

Strains and plasmids

All C. glabrataand bacterial strains and plasmids used in this study are listed in Table 2.1

Centre for DNA Fingerprinting and Diagnostics Hyderabad, India

Maruti Nandan Rai

A study of host-pathogen interaction in Candida glabrata

The immunoblots were quantified by densitometry software ImageJ 1.17 developed by Wayne Rasband, NIH Bethesda, MD (http://rsb.info.nih.gov/nih-image).All experiments were done at least in triplicates and results were expressed as mean ±s.e.m. A two tailedStudent’s t-test was done in Graph pad to arrive at p values and differences were considered statistically significant when p-value was less than 0.05 (*p≤ 0.05), highly significant(**p≤ 0.01)andextremely significant (***p≤ 0.001)

Quantification of blots and statistical analysis

competent cells pre-inoculum was prepared. A single bacterial colony was picked from LB agar plate that has been incubated for 16-20 hours at 37 °C and inoculated into 3 mlLB medium and incubated overnight at 37 °C temperature with 200 rpm shaking. 1% of this pre-inoculum was sub cultured in 100 ml LB-broth and incubated at 18 °C until OD 600 reached 0.5 -0.6 (approx.). Culture was kept on ice for 10 min. with constant shaking. Cells were pelleted by centrifugation at 2000xg/4°C/8 min. Pellet was resuspended in 40 ml of ice-cold Innoue buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000 xg/4°C/8 min. Pellet was resuspended in 8 ml of TB buffer inwhich final concentration of DMSO was 7% and left on ice for 10 min. 100μl aliquots were made and snap frozenin liquid nitrogen and stored at -80 °C

All the salts (10 mM PIPES, 15 mM CaCl2.2H2O, 250 mM KCl,55 mM MnCl2. 2H2O) except MnCl2were dissolved in water and pH was adjusted to 6.7 with 1N KOH. MnCl2was dissolved separately in water. MnCl2was added drop wise while stirring (MnCl2if added directly will give a brown colour to the solution and precipitates;hence it needs to be dissolved separately). Solution was then sterilized by filteringand stored. To prepare

PreparationofUltra competent cells

Of theligation mixture,2μl (of total volume of 10 μl reaction)was added to atube of 100μlultra competent DH5α bacterial cells and incubated in ice for 30 minutes. The tubewas quickly transferred to a water bath maintained at 42°C to give a heat shock for 90 seconds and again quickly transferred to ice. 1ml of LB broth was added to the tube and then incubated at 37°C for 1 hour. The bacterial cells were then pelletdownby centrifugation at 6000 rpm for 5 minutes and plated on LB agarcontaining appropriate antibiotic

Transformation of ligated DNA

A total of 100-200ng of DNA was used in each ligation reaction. Vector to insert ratio of 1:3 to 1:5was maintained. The reaction volume was generally maintained at 10μl containing 1μl of 10X ligation buffer (provided by the manufacturer) and 0.05 Weissunit of T4-DNA ligase. The reaction was carried outat 16ºC for 14-to 16-hrs or at room temperature for 4hours

Ligation of DNA

Digested DNA fragments required for ligation were eluted from the agarose gel after electrophoresis. The gel was visualised over a UV illuminator and section of it containing the desired DNA fragment was carefully sliced out from the gel. The sliced agarose gel was then processed using commercially available gel elution kitsfor this purpose. The elution efficiency was checked by running a small aliquot of DNA sample on agarose gel

Purification of DNA by gel elution

For restriction digestion(either single or double), 0.5 to 1μg of DNA was used in a reaction containing2 to 5units of commercially available restriction enzyme(s)and 5μl of the recommended buffer (suppliedas 10X concentrationsby the vendor)in atotal reaction volume of 50μl. The reaction mixture was incubated for 2 h or overnight at 37°C. The digested DNA fragments were then visualised by ethidium bromide staining after electrophoresis on agarose gels. Commercially available DNA size markers were loaded along with the samples to ascertain or estimate the sizes of the digestedfragments

Restriction enzyme digestion

Overnight grown bacterial culture (3ml)was pellet down by centrifugationat4ºC for10-min at 6000 rpm. The cells were re-suspended in 200μl of Resuspension solution(solutionI). 400μl of freshly prepared Lysissolution(solution II)was then added and mixed by gently inverting the tubesfor 4-6 times and allowed to lyse for 5 min at room temperature.The complete lysis was ascertained by uniformity and clarityof the contents. Subsequently, 400μl of Neutralization solution(solution III)was added and the tubes were inverted 4-6 timesand gently for homogeneous mixing followed byincubation for 5 min on ice. After centrifuging at 12,000 rpm for 15-min, supernatant was decanted into a fresh tube, and0.7 volume of iso-propanol was added.Theprecipitated nucleic acids were then recovered by centrifugation at 12,000 rpm for 30-min. The pellet was washed once with 70% ethanol, air-dried and re-suspended in 100μl of TE-buffer. It was treated with RNase at a concentration of 20μg/ml by incubating at 37ºC for 1hour. It was further extracted with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) mixture. After centrifugation, the clear supernatant was used for recovering the nucleic acids. The nucleic acids were precipitated with 2.5 volumesof ethanolin presence of3 M sodium acetate. In case where high purity plasmid preparations are required (for transfection to cells) the plasmid isolation was carried out with the commercially available midiprep or miniprep kits following the manufacturer’s instruction. Plasmids were observed on 1% agarose gel

Isolation of plasmid DNA

Recombinant DNA techniques

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits from Perkin-Elmer on an automated sequencer (model 377, Applied Biosystems), following the manufacturer’s instructions

DNA sequencing

Agarose gels were preparedby boiling appropriate amount of agarose in TAEbuffer. After dissolution, it was cooled and then poured in a casting tray containing a comb for desired number of wells. The gel was allowed to solidify and then shifted to horizontal electrophoresis tank containing TAE buffer. The DNA samples were mixed with appropriate volumes of 6X DNA loading dye, loaded on the gel andelectrophoresedat appropriate voltage and current conditions (generally 80 V,400 mA). The gel was stained in ethidium bromide solution(1 μg/ml)for 15-min at room temperature and visualisedby fluorescence under UV-light in a UV-transilluminator

Agarose Gel Electrophoresis

2μg of total RNA was reverse-transcribed using SuperScript III Reverse Transcriptase which is a commercially available version of M-MLVRT with reduced RNase H activity and increased thermal stability.According to manufacturer’s protocol1μg of RNA,1μl oligo(dT)(500ng),1μl 10mM dNTPand nuclease freewater was added to afinal volume of 13μlin a PCR tube.Thismixture was then incubated at 65°C for 5 minutesin a thermo cyclerand then quicklytransferredtoicefor 1minute. To this 4μl of 5X first strand buffer 1μl of 0.1MDTTand1μl ofRNaseOUT (40U/μl) were added. Then contents were then mixed and 1μl (200 units/μl) of SuperScript III RT was added. Themixture was then incubated at 50°C for 60 minutesin a thermo cycler.Lastlythe reaction was stopped byincubating the mixture at 70°C for 15 minutes. The cDNA thus prepared was then usedas a template for PCR

RT-PCR (Reverse Transcriptase PCR)

The quantity and purity of nucleic acids was determined by measuring the absorbance at 260 and 280 nm. The concentration of nucleic acids was calculated by considering the OD (λ260)= 1 corresponding to50μg/ml DNA and 40 μg/ml ofRNA. The purity of nucleic acids was checked by their A260/A280 ratioconsidering 1.8 for DNA and 2.0 for RNA. These measurements were done in NanoDrop 2000 UV-Vis Spectrophotometer

Quantification of nucleic acids

Total RNA was isolated by TRIzol method using the manufacturer’s protocol. Briefly, medium was removed from culture dish and recommended amount of TRIzol wasadded directly on to the dish and kept at room temperature for 5 minutes for lysis of cells. The cellular homogenate was then transferred to a 1.5ml microcentrifuge tube. For each mlof TRIzol, 200μl of chloroform was added and tubes were shaken vigorously for 10 seconds to completely dissociate the nucleoprotein complexes, followed by vortexing for about 30 seconds. The mixture was kept for 3-5 minutes at room temperature and then centrifuged at maximum speed of 12,000 rpm for 10 minutes. The upper aqueous phase was transferred into a fresh micro centrifuge tube and RNA was precipitated by adding 500μl of iso-propanol. The RNA pellet was obtainedby centrifugation at 12,000 rpm for 30 minutes at 4°C. The pellet was washed with 1ml of chilled 70% ethanol followed by centrifugation at 12,000 rpmfor 5minutes. The supernatant was removed and the pellet air-dried for about 5 minutes. The pellet was resuspendedin 30-50μl RNase free deionisedwater and dissolved at 55ºC followed by quantificationusingnanodrop spectrophotometerfor further use.The RNA integrity was checked by evaluating the 18S and 28S rRNA signals by running 1μl of total RNA on denaturing agarose gel stained with ethidium bromide

Total RNA isolation from cultured cells

Molecular techniques

nvolved use of GFP based vector system, the expression of the transgene was visualized under fluorescent microscope with excitation filter of 485+20 nm

Transient transfection of plasmid DNA in cellswas performed usingLipofectamine 2000transfection reagentaccording to manufacturer’s protocol. Briefly, 0.5 to 1million cellswere seeded in a 35mm tissue culture dish one day prior totransfection. For each 35mm dish, 4μg DNA was mixed in 250μl of Opti-MEMin one polypropylene tube. In another tube 10μl of Lipofectamine 2000 was diluted in250μl Opti-MEM and incubated at room temperature for 5 minutes. DNA and Lipofectamine 2000 were mixed together and allowed to form complexes for 30minutes at room temperature. Meanwhile, the adherent cells were washed twicewithPBS and 1ml of Opti-MEM was added. 500μl of complexes were then added to each dishcontaining cells and medium. After 6-8 hrs, the medium containing complexes wasremoved and complete medium was added and transgene expression was evaluated 24-48 hrs after transfection. Since most of the experiments

Transient transfection in adherent cells

Themixture is incubated in a water bath at 37⁰C for 15 min and afterwards transferred on ice and 4μl of DNA loading buffer is added. The samples were then run on a polyacrylamide gel electrophoresis which had been pre-run for 30 min. Electrophoresis was carried out at 4⁰C for 3h till the bromophenol blue migrated to 2cm above the bottom of gel. The gel was taken out and kept on Whatman filter paper sheet and covered by saran wrap followed by drying in a gel dryer at 80⁰C for 1h under suction. The dried gel was exposed to phosphoimager screen by keeping in phosphoimager cassette overnight

A binding reaction mixture was prepared by adding the following components to a microcentrifuge tube on ic

Binding reaction

The reaction was carried out by incubating at 37⁰C for 30 min. The reaction was stopped by adding 2μl of 0.5M EDTA, pH 8.0 and keeping on ice. A spin column was prepared using 1ml syringe and packed with sterile Sephadex G50 slurry and reaction mixture is applied on the top. The eluate is collected in different microcentrifuge tubes and radioactivity was counted using Geiger counter. The tube showing 7 to 9X106was used for experiment. The column containing the unincorporated [γ-32P] ATP was discarded in radioactive waste bin. The radiolabelled oligonucleotides were annealed with their corresponding complementary unlabelled oligonucleotides. A 50 fold molar excess of the latter was used for annealing for conversion of labelled single strand to double strand. Thetubes were kept in boiling waterbath for 3 min followed by room temperature for 30 min. The tubes were transferred to ice and the oligonucleotides were diluted to 4fmoles/μl using sterile H2O

The oligonucleotides were labelled at their 5'end with 32P using T4 polynucleotide kinase (T4 PNK) enzyme in a reaction given belo

end labelling of the oligonucleotides

Electrophoretic mobility shift assay

Adherent cells growing either on cover slips or chamber slides were fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were washed with PBS thrice for 5 min each and blocking was done in 2% BSA(preparedin PBScontaining 0.3% Triton-X 100) for 1h.The cells were incubatedwith primary antibody(dilutedin PBScontaining 0.3% Triton-X 100)for 2h at room temperature or overnight at 4⁰C.The cells were washed with PBS thrice for 5 min each followed by incubation withAlexa Fluor 488-or 594-conjugatedsecondary (anti-mouse/rabbit) antibodiesfor 1h. Then the cells were mounted on microscopicslides using Vectashieldmountingmediumcontaining nuclear dye DAPI. Imaging was done byeither the laser scanning confocal LSM510 or LSM 750 (Carl Zeiss, Oberkochen, Germany) or fluorescence inverted (Olympus 1X51, Tokyo, Japan) microscope

Immunofluorescence Microscopy

Equal amount of proteins were loadedon an appropriate percentageof denaturing SDS-PAGE gel. After completion ofthe run, the gel was over laid on a PVDF membranecut to the size of gel and sandwiched between filter paper sheets and kept inthe blotting cassette in the presence of transferbuffer. Finally the cassette was put in themini transblotapparatus and blotting was done for 2-3hours at a constantvoltage of 80Vat 4⁰C. For blocking the nonspecific sitesmembrane was incubated with blocking solution(5% non-fat milk solution in TBST)with gentle shaking for 1 hourat room temperature. Excess milk from the membrane was washedoff with TBST and themembrane was incubatedwith primary antibody diluted in 1XTBST for 3 hours atroom temperature or overnight at 4°C withshaking. After incubation the membrane was washedwith TBST and incubatedwithappropriate secondary antibody (conjugated with horse-radish peroxidase)diluted in5% fat free milk solution (in TBST) for 1hat room temperature.The blotwas later washed thricefor 10min eachwith TBST and processed for the detection of proteinsignal using ECL-prime chemiluminescencedetection reagent followed by detectionof signal either on X-ray filmor in a chemiluminescence detectionsystem(Proteinsimple, California, USA)

Immunoblotting

BCA (Bicinchoninic acid) method was used to determine the proteinconcentrationin various samples. The Cu2+ions from cupric sulphate (present inBCA reagent B) reagent arereduced to Cu+by the protein in an alkaline medium. The cuprous ion (Cu+) then combines with BCA (present in BCA reagent A) to give a purple colour whose intensity is proportional to the amount of protein present in the samples. This intensity is measuredby colorimetry at 562 nm. BCA reagent was prepared by mixing reagent A with reagent B in avolumeratio of 50:1. A standard curve was generated using increasing concentrations of BSA (2-10μg) in a 25μl reaction, in a 96 well plate. Cell lysates were also dilutedto same volume in parallel wells. 200μl of BCA reagent was then added to each well and incubated at 370C for 30 minutes. The absorbance readings were then takenin a spectrophotometer at 562 nm. Total protein was quantified by calculation of the slopes of regression lines ofabsorbanceand BSA standards

Protein estimation

For preparation ofcellular homogenate from adherent cell culture, the medium was first removed and cells were washed with ice cold 1X PBS. The cells were then scraped in 1X PBS and pellet down by gentle centrifugation (4000 rpm for 2 minutes) at 40C. Cell lysis buffer was then added to the cell pellets and lysis was allowed for 30 minutes on a rotor at 4⁰C. Post lysis, cellswere centrifuged at 13000 rpm for 10min at 4°C. The pellet was discarded and supernatantwascollectedas cell homogenate

Extraction of total cellular protein

drop wiseaddition and kept at 4⁰C for 24h. Cells were then washed with PBS and stained with DNA staining solutionat 370C in darkwith intermittent shaking. The DNA content of cells was measured by flow cytometryusing FACS-Aria (Beckton-Dickenson) at 695 ±20 nm using a 655 long pass filter.The DNA content was then analysed by FACSDivaor FlowJosoftwareto evaluate the various phases of cell cycle. The diploid 2N DNA content was referred as G1/G0 population and the 4N DNA content was referred as G2/M population. Cells with intermediary DNA content (between 2N -4N) content were considered as S phasecells and those below 2N DNA content as sub G0 cells

Thecells were collected at various time points by trypsinization, washed in phosphate buffered saline (PBS, pH 7.2) and fixed in chilled 70% methanol: ethanol (1:1) solution by

Cell cycle analysis

Cells were seeded in replicates of five @ 3X103cells per wellinfive different 96well cell culture platesand grown in complete media. The method described earlier was slightly modified and followed (Gillies et al., 1986). After every 24h of seeding, one plate was stained with 0.2% crystal violet in 2% ethanolfor 15 minutestill 4thday i.e. 96h.One plate was stained just after the cells get attached to use as 0h time point. Excess dye was removed from the plates by washing with ample amount of water. Crystal violet dye incorporated in the cells was extracted using 0.1% SDS solution by shaking for 10 minutes on a shaker. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. The experiment was repeated at least three times and the average absorbance was plotted for each time point to generate a growth curve

Cell growth Assay

Table2.2: Cell types used in the present study

In the present thesis, various cell types were used for which the details are provided in the Table 2.2. SiHa, HeLa, HaCaT, U2OS, SaOs , A549,HPLD andHEK-293cells were grown in Dulbecco’s modified Eagle’s medium (HyClone, Thermo Scientific, Logan, Utah, USA) supplemented with 2 mM glutamine (Gibco BRL), 100 U/ml penicillin and streptomycin (Gibco BRL, Carlsbad, CA, USA), and 10% fetal bovineserum (Gibco BRL, Carlsbad, CA, USA) under humified conditions at 37°C and 5% CO2.Cells were grown in cell culture dishes till they attained 70% confluency. For sub culturing, these were then trypsinised using 0.05% Trypsin EDTA solution and incubated for 5 minutes at 370C for cells to be detached from surface. The detached cells were then collected by gentle tapping the dish and pipetting. Trypsin was then inactivated by addition of FBS containing culture medium, transferred to a 15 ml tube and centrifuged at 1500 rpm for 2 minutes in a hanging bucket centrifuge. The cell pellet was then resuspended in complete medium and counted in Neubauercell counting chamber. Viability of the cells was checked by trypan blue exclusion method.Appropriate number of cells wasthen sub cultured in fresh cell culture dishes with culture medium as per the experimental requirements

Maintenance of cell lines

Cell biology methods

Malachite green reagent

Reaction Buffer

Cell lysis Buffer

Calcineurin phosphatase assay

DNA staining solution

Fixative

For cell cycle analysis by flow cytometry

Inoue buffer

For preparation of Ultra competent cells

DNA loading dye

Agarose gel

TAE

For DNA electrophoresis

Neutralization solution(Solution III)

Lysissolution(Solution II)

Resuspension solution(Solution I)

For Plasmid isolation

Binding Buffer (10X)

EMSA Buffer

Nuclear lysis buffer

Polydeoxy (Inosinate-cytidylate) (Poly dI-dC)

For Electrophoretic mobility shift assay (EMSA)

Nuclear extractionbuffer (without protease inhibitors)

Cytoplasmic extractionbuffer (without protease inhibitors)

For Cell fractionation

Blocking buffer: 2% BSA

Permeabilization buffer: 0.2% Triton X100

Fixative : 4% Formaldehyde

For Immunofluorescence

Stripping Buffer

Blocking Buffer

TBST

Transfer Buffer

Running Buffer

Stacking and resolving AcrylamidegelsResolving gel (10 ml)

6X protein loading buffer (Laemmlibuffer)

Cell lysis buffer(RIPA Buffer)

For Immunoblotting

Tris Buffered Saline (TBS)

Phosphate Buffered Saline (PBS)

General Buffers

Ammonium persulfate(APS)

Acrylamide (29:1)

Phenylmethylsulfonyl fluoride (PMSF)

Benzamidine

Aprotinin

Leupeptin

NP-40ComponentsFinal concentrationFor 10 mlNP-4010%1mlH2O9ml

Dithiothreitol (DTT)ComponentsFinal concentrationFor 5 mlDTT1.0M0.7725gH2Oq.s

Ethylenediamine tetraacetic acid (EDTA), pH 8.0ComponentsFinal concentrationFor 500 mlEDTA0.5M93.05gH2Oq.sThe pH is adjusted to 8.0 using 10M NaOH

Ethylene Glycol Tetraacetic acid (EGTA), pH 7.0ComponentsFinal concentrationFor 50 mlEGTA0.1M1.902gH2Oq.sThe pH is adjusted to 7.0 using 10M NaOH

Potassium Chloride (KCl)ComponentsFinal concentrationFor 100 mlKCl2M14.91gH2Oq.s

Sodium Chloride (NaCl)ComponentsFinal concentrationFor 100 mlNaCl5M29.22gH2Oq.s

Potassium Chloride (KCl)

HEPES pH 7.9ComponentsFinal concentrationFor 100 mlHEPES1M23.83gH2Oq.sThe pH wasadjusted to 7.9 using 10M NaOH

Stock solution

Buffers and solutions

forpreparation ofregular buffers and solutions viz. Tris, Glycine, SDS, Sodium Chloride, Potassium Chloride, Disodium Phosphate,NP-40, Tween 20, TritonX100, Formaldehyde, Glycerol, Agarose, Acrylamide,Bis-Acrylamide,Ammonium per sulphate (APS), TEMED,BSA, Propidium Iodide, RNase Aetc. were obtained from Sigma(St Louis, MO, USA). PVDF membrane, X –ray films and western blotting detection reagent (ECL prime) were obtained from GE Healthcare (Little Chalfont, UK). Proteaseinhibitor tablets were obtained from Roche (Penzberg,Germany). Anti mouse and anti-rabbit secondary antibodies tagged to HRP (Horse radish peroxidise) were obtained from Bangalore Genei(Peenya, India). Secondary antibodies for Immunofluorescence (anti mouseIgGand anti rabbitIgG) conjugated to Alexa Fluor (488 and 594) from Molecular Probes, Invitrogen and Vectashield mounting medium with DAPI wasobtained from vector laboratories(Burlingame, CA, U.S.A).Antibodies from different sources were used in the present study. The list of different antibodies used in the present thesis is provided in Table 2.1.Table 2.1: List of antibodies used

Media for cell culture (DMEM and Ham’s F12) and foetal bovine serum (FBS) were obtained from Gibco, Invitrogen (Carlsbad, CA, USA). Cell culturereagents such asTrypsin, Phosphate Bufferedsaline (PBS), Antibiotics, Glutamine, etc. were also obtained from Gibco, Invitrogen (Carlsbad, CA, USA). Chemicals for cell culture experiments Aphidicholin, Nocadazole, Polybrene, and Puromycinwere obtained from Sigma (St Louis, MO, USA). Cyclosporine A, MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), wortmannin, UO126, SP 600125, cycloheximide, camptothecin, Tacrolimus/FK506 , Tween 20 and Malachite green were obtained from Sigma-Aldrich (St. Louis, MO, USA). Specific calcineurin substrate RII peptide, calmodulin, eIF-2α inhibitor salubrinal, MG-132 and caspase inhibitor z-VAD FMK were obtained from Calbiochem (San Diego, CA, USA). Cytotoxicity detection kit (LDH) was obtained from Roche Diagnostics, (Mannheim, Germany).Live /Dead cytotoxicity assay kit was obtained from Molecular probes, Life technologies, USA.Lipofectamine-2000 and Opti-MEM for transient transfections were also obtained from Invitrogen(Carlsbad, CA, USA).Growth media for bacteria (LB) was obtained from HiMedia laboratories (Mumbai,India). Enzymes used for recombinant DNA experiments (Restriction endonucleases, DNA ligase) were obtained from New England Biolabs (Ipswich, MA, USA). Markers for DNA and protein gels were from Fermentas (Vilnius, Lithuania). Various kits used for macromolecular isolation (Plasmid isolation kit-Mini and midi, Gel extraction kit, PCR purification kit, RNA isolation kit) were procured from Qiagen(Hilden, Germany) or HiMedia (India).Trizol reagent for RNA isolation was obtained from Invitrogen (Carlsbad, CA, USA). BCA protein estimation kit was from Pierce (Rockford Illinois, USA). Cell fractionation kit was obtained from Fermentas (USA). Kitfor TUNEL assay kit wasobtained from Invitrogen(Carlsbad, CA, USA). PCR reagents (PCR buffer, dNTPs, MgCl2, Taq DNA polymerase) were obtained from Fermentas. Polymerasefor long PCRs (AccuTaq) was obtained from Sigma. Reverse transcriptase (SuperScript III) was obtained from Invitrogen. Various chemicals required

Media, reagents, chemicals and antibodies

Studies on Calcineurin

toManipal

Studies on Calcineurin-NFAT signaling in cellular proliferation and effect of its inhibitor, cyclosporine A, in cell death response

All data were presented as mean ± standard deviation (SD)/standard error of mean (SEM) fromthree independent experiments. Statistical analysis was performed using Student's t-test and one-way ANOVA followed by a post hoc Tukey testwherever applicable. Results were analyzed and illustrated by SPSS statistical software package (SPSS for Windows,version 16). Comparisons are done within and between the test groups (i.e.,parentalcells and profilin-stable cells). Asterisk (*) symbol indicates statistical difference between parentaland profilin-stable cells, whereas Number (#) and Dollar ($) signsindicatestatistical difference within parentaland profilin-stable cells, respectively. Significance of results was determined as p ≤ 0.01 and p ≤ 0.05(*indicates p ≤0.05, **indicates p ≤0.01, *** indicates p ≤0.001 and **** indicates p ≤0.0001

Statistical analysis

AutoDock tools 1.5.6 (Morris et al., 2009) and PyMOL (Sanner, 1999) was used to prepareand analyze the docking simulations.Preparation of protein structures for docking:The three dimensional structure of PTEN (PDB ID: 2PBD) (Lee et al., 1999)and profilin-1 (PDB ID: 1D5R) (Ferron et al., 2007)were obtained from protein data bank, PDB (www.rcsb.org).Prior to initiating the docking simulations, all non-protein molecules were removed from the intact PTEN and only the chain P was retained. In the same way, we retained (‘chain A’) for profilin-1. All the non-protein molecules were removed usingChimera(Pettersen et al., 2014).Protein PTEN was used as receptor and profilin-1was used as ligand. Kollman united atom charges and polar hydrogen were added to the receptor protein. Receptor protein was kept rigid in docking process, assuming there is no induced conformational change upon ligand binding. In contrast to the protein, torsional flexibility was permitted for the ligands via the side-groups and backbone of protein was kept rigid.Atomic affinity and electrostatic potentials were computed for a grid boxand positioned around the approximate centre of the binding site. The grid box size was set at 58.0 X 76.0 X 58.0 Å (x, y, and z) with the spacing between grid points at 1 Å and the center at 36.253 X 82.395 X 31.728 for x, y and z coordinates, respectively. All other docking parameters remained as the Autodock default settings.Analysis of docking data:AutoDockVina (Trott and Olson, 2010) software was used for docking simulations. The resulting docked conformations were clustered into families of similar conformations, with the root mean square deviation (r.m.s.d.) clustering tolerance of 2.0 A ̊. As a rule, the lowest docking-energy conformations were included in the largest cluster. The process was repeated for each remaining conformation, until all the conformations belonged to a single cluster. A new reference conformation was defined every time a new cluster was created. Following this, the best-docked conformation is selected as the lowest energy pose in the most populated cluster, i.e., the cluster with the highest convergence out of the 100 trials. This differs from the practice of simply choosing the overall lowest energy pose. Thus, we avoid picking lower energy ranked poses belonging to sparsely populated clusters, which can be considered as ‘chance-hits’ given that the docking algorithm was unable to converge to similar poses in other independent trials

Molecular docking

The highly efficient Ultra competent DH5α cells for transformation were prepared by Inoue method (Inoue et al., 1990). Briefly, a single bacterial colony was picked from LB agar plate, inoculated into 10 ml LB medium and incubated overnight at 37°C temperature with 200 rpm shaking. Following day, 1% of the pre-inoculum was sub-cultured in 100 ml LB-broth and incubated at 18°C until OD600 of 0.5-0.6 was reached. Culture was then kept on ice for 10 min with constant shaking. Cells were pelleted by centrifugation at 5000 rpm at 4°C for 10 min. Pellet was resuspended in 40 ml of ice-cold Inoue buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000g at 4°C for 10 min. Pellet was resuspended in 8 ml of TB bufferin which 560 μl DMSO was added and left on ice for 10 min. Finally, 100 μl aliquots were made by snap freezing in liquid nitrogen and stored at -80°C

Preparation of Ultra competent cells

In a typical ligation reaction, a total of 100 ng of vectorwas usedwhereas;theconcentration of insert varies from 300 to 500 ng. For10μlreaction volume, 1μlligation buffer (provided by the manufacturer) and 0.5μlof T4-DNA ligasewere added in vector-insertmixture. The reaction was maintainedat 16oC for 14-16h.After ligation, 2μl of the ligation mixture (of total volume of10 μl reaction) was added to a vial of ultra-competentDH5α bacterial cells and incubated in ice for 30 minutes. The ligation mixture was allowed toheat shock at 42oC for 90 seconds followed by quick transfer onice. About1ml of LB broth was added to the tube and incubated at 37oC for 1 hour. The bacterial cells were then pelleted by centrifugation at 6000rpm for 5 min and plated on LB plate containing appropriate antibiotic

Ligationand transformationof DNA

For construction of desired clones, 1-2 μg of DNA was used for restriction digestion. In a typical reaction, 2-5units of restrictionenzymeswereused in the total reaction volume of 50μl along with5μl ofrecommended buffer (supplied as 10X digestion buffer). The reaction mixture was incubated for 3-5 hat 37oC. The digested DNAwasthenloaded along with DNA size marker and separated on agarose gel electrophoresis. The gel was visualized over a UV illuminator and section containing the desired DNA fragment was carefully sliced out. The sliced agarose gel was then processed using commercially available Gel Extraction kit (Qiagens) for this purpose

Digestion and elution of DNA

Plasmid DNA was isolated from10-100ml of bacterial culture grown overnight by using commercially available Miniprep or Midiprep kits (Invitrogen) as per manufacturers’protocol. Briefly, bacterial cells werepelleted by centrifugation at 6000 rpmfor10 min.The pelleted cells werere-suspended in 300μl-4ml of resuspensionsolution containing RNase H.About300μl-4ml of alkaline lysis solutionwas then added and mixed by gently inverting the tubesuntil clear and uniform lysatewas appeared. Subsequently, 350μl-5mlof neutralizing solutionwas added,the tubes were inverted repeatedly and gently to ensurehomogeneous mixing, followed by incubation for 5 min on ice. After centrifuging at 12,000 rpm for 15 min, supernatant was passed through commercially supplied filter columns by either gravity flow or high-speedcentrifugation. During this step, plasmid DNA binds to the column and was recovered by passing elution buffer through the column. The plasmid DNA present in the elution buffer was collected into a fresh tube and 70%(w/v)of iso-propanol was added.Theprecipitated nucleic acids were then recovered by centrifugation at 12,000 rpm for 30 min. The pellet was washed once with 70% ethanol, air-dried and re-suspended in desired amountof TE-buffer. Finally, the purity of plasmid wasobserved on 1% agarose gel

Isolation of plasmid DNA

DNA sequencing

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits from Perkin-Elmer on an automated sequencer (model 377, Applied Biosystems), following the manufacturer’s instructions

For preparing agarose gels,appropriate amount of agarose(0.8-2%) was dissolved by boilingin TAEbuffer, until clear slurry was formed.It wasthen poured in a casting tray containing a comb for desired number of wells. The gel was allowedto solidify andshifted to horizontal electrophoresis tank containing TAE bufferwith 1 μg/ml ethidium bromide. Appropriate volumesof 6X DNA loading dye were added in the samples and subjected to electrophoresis atconstant voltage (generallyat80 V), along with appropriate DNA ladder,untilband were resolved. The gel was visualised underUV-light in a Gel-Doc or UV-transilluminator

Agarose gel electrophoresis

The quantityand purity of nucleic acids weredetermined by measuring the absorbance at 260 and 280 nmusing automated NanoDrop instrument. The concentration of nucleic acids was calculated by taking 1 OD at 260nm = 50μg/ml for DNA and 40 μg/ml for RNA. The purity of nucleic acids was checked by their A260/A280 ratio

Spectrophotometric estimation of nucleic acids

The reaction mixture was then placed on a thermo-cycler with the required cycling conditions for amplification of the desired gene, as described in Table 2.7. The amplified products were purified by PCR purification kit (Qiagen) and either quantified by spectrometry or separatedby agarose gel electrophoresis.Table2.7:Cycling conditions for PCR

The PCR amplification of desired genes werecarried out using Taq polymerase reaction kit obtained from Fermentas.Typically, a reaction mixture containing primers with plasmid containing geneof interest or c-DNA was prepared as described in Table 2.6. Table2.6: Various components of PCR reaction mix

Polymerase Chain Reaction (PCR)

Total RNA, isolated by TRIzol method was reverse transcribed into cDNA by One step Access RT-PCR kit (Promega, Madison, WI), as per manufacturer’sprotocol.Briefly, 1 μg of RNA, 1 μloligo-dT(500ng), 1 μl 10 mMdNTP and nuclease free water was added in a PCR tube. This reaction mixture was incubated at 65°C for 5 minutes in a thermo cyclerand then quickly transferred to ice. To the mixture, 4μl 5X firststrand buffer, 1μl 0.1M DTT,1μlRNase-OUT (40U/μl) and 1 μl (200 units/μl) of SuperScriptIII were added.The contents were mixed by gentle vortexing and incubated at 37°C for 60 minutes in a thermo cycler. Finally,the reaction was stopped by increasing the temperature to72°C for 10minuteson thermo cycler. The cDNA thus prepared, wasused as a template for PCR.PCR was then performed for either amplification of the gene of interest or relative expression of desired genes by the using gene specific primers(Table 2.5). Products were separated by agarose gel electrophoresis (2%) and visualized by ethidium bromide staining.Table2.5: List of RT-PCR primersused in the study

Reverse transcriptase (RT)-PCR

Total RNA was isolated from the treated cells usingTRIzol method, essentially described by Donald and his co-worker (Donald et al., 2010).TRIzol is a single-phase solution of guanidinium isothiocyanate and phenolthat can concomitantly denature proteins and other biological material. Addition of chloroform to this leads to phase separation: proteins remains in organic phase whereas, DNA and RNA resolves to interphase and aqueous phase, respectively. Before starting the experiment, area was sanitized with RNAZap toremove any contamination of DNases. After treatment, culture media was gently removed from the dish without disturbing the cell monolayer. TRIzolreagentwas added directly on to the dish andcellswere allowed tosuspend in it by repeated pipetting. The cellular homogenate was then transferred into amicrofuge tubes. For each ml of TRIzolused, 200μl of chloroform was added andvortexedfor about 30 seconds, followed bycentrifugation at maximum speed of 13,000 rpm for 10 minutes. The upper aqueous phase wastransferred into a fresh micro-centrifuge tube and 500μl of ice-cold iso-propanol was addedto precipitate RNA.The RNA was pelleted by centrifugation at 13,000 rpm for 30 minutes at 4°C. The supernatant was decanted and the pelletwas allowed towash with 1ml of ice-cold 70% ethanol followed by centrifugation at maximum speed for 10 minutes. Finally, the supernatant was removed and the pellet was allowed toair-dryfor about 5-10 minutes and solubilized in 50μl RNase free deionized (DEPC-treated Milli-Q) water and quantified by spectrophotometry for further use

Isolationof total RNAfrom cultured cells

Table 2.4: List of double-stranded oligonucleotides used in the present study

The DNA-protein complexes were then separated from free oligonucleotides on 6.6% native PAGE gel. The samples were loaded into a native PAGE gel, which was pre-run at constant current (40-50 mA) for 15-30 minutes. Electrophoresis was performed at constant current (80-100 mA), till the bromophenol blue dye front reached 1-2 cm from bottom of the gel. The glass plate was carefully removed without disturbing the gel and the Whatmann filter paper no. 3, cut to the size of the gel, and wasplaced over it. The paper was pressed gently and the gel, which was now firmly stuck on the paper, was covered with saran wrap and kept for vacuum drying on the gel-dryer at 80oC for 1 h. After drying, gel was exposed on a Phospho-imager screen for 12-24 h and scanned on Phospho-imager to detect the band of interest.To determine the specificity of the transcription factor binding or sub-unit interacting to the desired oligonucleotide, super-shift assay was performed. For this, 8-10 μg of nuclear extracts were first incubated with desired antibodies (concentration varies for different) or their isotype control for 1h at 25°C, followed by incubation with binding reaction mixture. The various oligonucleotide sequences used in the present study are listed in Table2.4below

For detection of protein-nucleic acid interaction, an electrophoretic mobility shift assay (EMSA) was conducted as described by Hellman and Fried (2007). Breifly, 8-10 μg of nuclear extract protein was incubated with binding reaction-mixture containing either 32P end-labeled double-stranded oligonucleotides (NF-κB, AP-1, p53 or SP-1) or unlabeled oligonucleotide as shown in table 2.3. The reaction mixture was incubated at 37°C for 45-60 min. After completion of reaction, 6μl of 6X DNA-loading dye was added and mixed well by gentle tapping.Table 2.3: Binding conditions for DNA-protein complexes in EMSA

Gel shift assay

The mixture was then incubated at 37oC for 45 min to radiolabel the oligonucleotide. Simultaneously, the Sephadex G-50 column was prepared in 1 ml syringe. The reaction mixture was loaded on the column and the eluting fractions were collected in the microfuge tube by loading 200 μl milli-Q water on the top of the column. After collecting 5-6 fractions, the tubes were analysed using a GM counter for the amount of radioactivity. The fractions having specific activity between 3.5-4.5 X 106cpm/pmoles were pooled. To this, 100 pM of the complimentary strand of the oligonucleotide was added and heated at 95°C for 5 minutes. The mixture was allowed to annealat room temperature for 1 h and further used in Gel shiftassay

The oligonucleotides of different transcription factors such as NF-B, AP-1, p53 and SP-1 were 5′-end labelled using radioactive γ32-ATP (obtained from BRIT, BARC, Mumbai, India) and Polynucleotide kinase, as per manufacturers’ protocol. The reaction mixture containing the different components, described in the table 2.2below was added in a microfuge tube.Table 2.2: Various components of oligonucleotide labeling reaction mixture

Radioactive labelling of oligonucleotides

and stained with ‘Live & Dead’ cell assay reagent (5 μM ethidium homodimer, 5 μM calcein-AM) for 30 min at room temperature. Red (as dead) and green (as live) cells were analyzed under a fluorescence microscope (Labophot-2, Nikon, Tokyo, Japan).For flow cytometry, cells were transiently transfected with either empty vector or various constructs. After 12 h, cells were treated with a combination of CHX (cycloheximide, 25 μg/ml) and TNF (5 nM) for 24 h. Cells were washed, trypsinised and then subjectedto flow cytometry (FACS Aria, BD Biosciences) using Live-Dead Cytotoxicity assay kit (Invitrogen). Live versus dead cells were analysed using FlowJo software

Cytotoxicity assay: The drug-induced cytotoxicity was measured by the 3-(4,5-Dimethylthiazolyl-2)-2,3-diphenyltetrazoliumbromide (MTT) assay as essentially described by Mosmann et al., 1983. Briefly, 5x104cells/well were seeded in 96-well plate. After 12 h, cells were treated in triplicates with different agents for different concentrations and time (in a final volume of 100μl). After completion of treatment, 25 μl of MTT solution (5 mg/ml in PBS) was then added and incubated for 2 h. The cytotoxicity was evaluated by uptake and cleavage of yellow MTT dye to purple formazan crystals by dehydrogenase activity in mitochondria of the living cells. Thereafter, 100 μl of extraction buffer (20% SDS in 50% dimethlylformamide) was added. After an overnight incubation at 37ºC, the absorbance at 570 nm was measured using 96-well multiscanner autoreader(Bio-Rad) with the extraction buffer as blank. Absorbance values were normalized to untreated cells and represented in percent cell viability for different concentrations or treatments.Determination of nuclear fragmentation: The morphology of live and dead cells was observed by staining the nucleus with DNA intercalating dye, propidium iodide (PI).Briefly, cells were treated with several apoptotic inducers for different concentration or time. Thereafter, cells werewashed with PBSand fixed in ice-cold 80% methanol for overnight at 4°C. Following day,cells were washed and suspended in 100 μl of PI solution (0.1% Triton, 0.2 mg/ml RNase A and 50 μg/ml PI in PBS) for 30 min in dark. Cells were then mounted on slides and viewed under fluorescence microscope (in 560 nm filter) to determine morphology of intact or fragmentednucleus.Live &dead assay: The cytotoxicity of various drugs was determined using the commercially available Live/Dead assay kit (Molecular Probes, Eugene, OR). Live cells have intact membraneand active cellular metabolism,which allow Calcien-AM to permeate inside and get cleaved into green fluorescent compound, Calcein (ex/em ~495 nm/~515 nm) due to intrinsic cellular esterase activity. On the other hand, Ethidium homodimer-1 (EthD-1) enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em ~495 nm/~635 nm). Hence, the cell viability can be assayed by either flow cytometry or fluorescence microscopy.For imaging, cells with different drugs treatments were washed with PBS

Assays for Apoptosis

Secretory alkaline phosphatase (SEAP) assay: For SEAP assay, the culture supernatant was analyzed for SEAP activity essentially as per the Clontech kit protocol (Palo Alto, CA). Briefly, cells were transiently co-transfected with Lipofectamine 2000 transfection reagent, 0.5 μg of required plasmid DNA(s) with the protein of interest or empty vector, 0.5 μg of reporter plasmid containing NF-κB binding site cloned upstream of heat-stable SEAP (designated asNF-κB-SEAP)and 0.5 μg of green fluorescence protein (GFP) expression plasmid (Clonetech) in Opti-MEM media.After 6 h of transfection, cells were washed and cultured for 12 h in complete media, followed by treatment with different inducers. GFP positive cells were then counted to ensure similar transfection efficiency. At the end of treatment, cell culture-conditioned medium was harvested and 25 μl of medium was mixed with 20 μl of 5X buffer (0.5 M Tris, pH 9 and 0.5% bovine serum albumin) in a total volume of 100 μl in a 96-well plate followed by incubation at 65°C for 30 min. The plate was chilled on ice for 2 min and 50 μl of 1 mM 4-methylumbelliferylphosphate (MUP, substrate) was added to each well and incubated at 37 °C for 2 h. The activity of SEAP was assayed on a 96-well fluorescent plate reader (Fluoroscan, Lab Systems, MA) with excitation set at 360 nm and emission at 460 nm. The average number (± SD) of relative fluorescent light units for each transfection was then determined and reported as fold activation with respect to empty SEAP-transfected cells.Luciferase (Luc) assay:The cell pellet was lysed and extract was analysed as per Promega kit protocol.Briefly, cells wereco-transfected with Lipofectamine with 0.5 μg of reporter plasmid containing p53 binding site cloned upstream of luciferase (designated as p53-luciferase) and 0.5 μg of GFP constructs. After 6 h of transfection, cells were washed and cultured for 12 h, followed by treatment with different inducers of apoptosis. GFP positive cells were then counted.Cellswere pelleted down and lysed using the lysis buffer. The samples were freeze-thawed twice by storing them at -70oC to ensure total lysis. The supernatant,obtained by centrifuging the same at 11,000 rpm for 2 min was transferred to a fresh tube. About 100 μl of the substrate (Firefly luciferin, Promega) was added to the supernatant and light emission wasmeasured in luminometer by using a delay time of 2 sec andread time of 10 sec.The values were calculated as fold of activation over vector-transfected value

Reporter gene transcription assays

Uniquitination assay was performed as described by Choo and Zhang, 2009. Ubiquitination is an enzymatic process of the covalent attachment of polypeptide ubiquitin on specific lysine residuesof protein, which is thendegraded by proteasome complex. MG132 (carbobenzoxy-Leu-Leu-Leucinal), a proteolytic activity inhibitor of proteasome complex, is widely used to assess the stability of protein in vivo. Briefly, parental and profilin-stable cells were treated with 10 μM MG132 for 6 h. The whole cell extracts prepared in NTEN lysis buffer were then subjected to immunoprecipitation with anti-ubiquitin antibody. The analysis of ubiquitination was performed by immunoblotting with anti-PTEN antibody

invivo Ubiquitination assay

Retroviral based system was used for silencing of PTEN. BOSC23 packaging cells were grown in 100 mm culture dishes upto 80-85% confluency. Retroviral RNA vector containing either scrambled control shRNA or pool of PTEN shRNA along with a PCL-Ampho helper plasmid were co-transfected using Lipofectamine 2000 reagent to generate viral particles. After 48 h, supernatant containing viral particles were used to infect MDA-MB-231 cells in the presence of polybrene (8g/ml). For p53 gene knockdown, TP53 mission shRNAs obtained from Sigma Aldrich (St Louis, MO, USA) were transfected using Lipofectamine 2000 (Invitrogen, USA) and non-targeting shRNAs (Sigma) were used as controls. The cellular homogenates were prepared 36-48 h post transfection and were subjected to immunoblotting to check the levels of protein knockdown

RNA interference

CHX pulse chase assay was performed as essentially described by Zhou (2004). Cycloheximide (CHX), a protein biosynthesis inhibitor was used to determine the half-life and stability of protein of interest. CHX blocks translation elongation step, thereby halting the synthesis of new proteins and therefore, time course degradation of protein can be studied. Briefly, parental and profilin-stable cells were seeded in 35 mm culture dishes and treated with CHX (50 μg/ml) the following day. Cells were harvested at different time points and level of protein was determined by immunoblotting

Cycloheximide (CHX) chase assay

Immunofluorescence assay was carried out as described by Bhattacharyyaet al., 2010.Adherent cells weregrown either on cover slips. After treatment, cells were fixed with 3.7% paraformaldehyde solution in PBS for 15 min and permeabilised with 0.5% Triton X-100 at room temperature for 10 min, followed by blockingin PBS containing 2% BSA for 1h. Post blocking, cells were incubated with a primary antibody in PBS (1:200 to 1:500) for 2 h. After washing, cells were incubated with fluorescent-conjugated secondary antibodyin PBS(Alexa Fluor 488 or 594 goat anti-rabbit or antii-mouse, 1:1000) for 30 min. After final wash with PBS, nuclei were counterstained with DAPI containing mounting medium (Vectashield, USA). All the steps were performed at room temperature, unless otherwise stated. Images were obtained using either the laser scanning confocal LSM510 (Carl Zeiss, Oberkochen, Germany) or fluorescence inverted (Olympus 1X51, Tokyo, Japan) microscopes

Immunofluorescence

Co-Immunoprecipitation assays were performed essentially as described by Lee (Lee, 2007). For a typical immunoprecipitation assay, cells were washed with ice-cold PBS and scapped in ice-cold microfuge tube. Then, cells were lysed with NETN buffer (containing 1 μg/ml each of leupeptin, aprotinin, 10mMeach ofNaF and phenylmethylsulfonyl fluoride (PMSF))on shaking rotator in cold room for 30 min. After centrifugation, the whole cell lysate (500 μg-1 mg) obtained was incubated with 1 μg of antibody of interest(orwithisotype control)on shaking rotator in cold room for 3 h, followed by addition of 10-20 μl of ProteinSepharose A/G beads (Santa Cruz) for 1 h. The immuno-complexes bound to beads were then pelleted atlow speed centrifugation (2500 rpm for 3 min) and washed three times with NETN buffer. The proteins bound to beadswere resolved by SDS-PAGE and immunoblotting was performed accordingto standard protocol described earlie