2.6.1 Cell culture, transfection, and viral infection
HEK293, U87MG, and HCT116 cells and their respective derivatives were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco) and penicillin/streptomycin (100 mg/mL, Hyclone), at 37°C in a CO2 incubator.
Transient transfection of HEK293 and cells was performed using the calcium phosphate technique, as previously described (F. L. Graham & van der Eb, 1973). Briefly, cells were seeded at low density in either 100 mm or 150 mm tissue culture dishes overnight. Cells were transfected with the indicated DNA plasmids for 16-24 hr. Fresh media was added to dishes, or cells were split into additional dishes, and incubated at 37°C overnight. In contrast, transient transfection of HCT116 cells was performed via a liposome-based method using PolyJet in vitro DNA transfection reagent, as described by SignaGen (2009). Murine PTEN plasmids were used to ensure expression of a Ser residue at the 398 position, normally a Thr residue in mammalian PTEN. All point mutants were generated by using the QuickChange site-directed mutagenesis kit (Agilent) or by traditional subcloning techniques.
U87MG and HCT116 PTEN-/- cells were engineered to stably express Flag-tagged murine wild-type PTEN or PTEN mutants via viral transduction with lentiviruses expressing pLVX-3xFLAG-mPTEN or mutants. Briefly, virus was produced by co-transfecting HEK293 cells at low density with the indicated lentiviral expression vectors and lentiviral packaging vectors, psPAX2 and pMD2.G for 24 hr. Virus was collected from supernatant and filtered (0.45 µM) 48 hr after transfection. Polybrene was added to increase infection efficiency. Receiver cells seeded at low density were infected for 24 hr, and then, fresh media was added. Subsequently, cells were
passaged and selected with hygromycin B (Invitrogen), ranging from 200 µg/mL to 300 µg/mL, for three to five days, dependent on survival of negative control cells.
2.6.2 Cell lysis and immunoprecipitation
Unless indicated otherwise, cells were lysed for 30 min on ice in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 5 mM NaF, 12.5 mM β-glycerophosphate), supplemented with fresh 0.1 mM sodium orthovanadate, 1 mM DTT, and a protease inhibitor cocktail (Sigma-Aldrich). Lysates were sonicated for 10 s and clarified by centrifugation at 15,000 x g for 15 min at 4ºC. Protein lysates were normalized for total protein content using Bradford reagent (Bio-Rad). Whole cell lysates were resuspended in 5x Laemmli loading buffer.
Immunoprecipitations were performed by incubating protein lysates with anti-FLAG M2 agarose beads (Sigma-Aldrich) for 1 hr or the indicated antibody overnight, gently rotating at 4ºC.
Protein lysates incubated with antibody overnight were subsequently incubated with protein A sepharose beads for 1 hr, gently rotating at 4ºC. Beads were washed three times with cold RIPA buffer and resuspended in 2x Laemmli loading buffer.
2.6.3 Subcellular fractionation
Cells were pelleted in phosphate buffered saline (PBS, Gibco) at 1,000 x g at 4ºC, and then, lysed for 5 min on ice in Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 340 mM sucrose, 10% glycerol), supplemented with fresh sodium fluoride, β-glycerophosphate, DTT, protease inhibitor cocktail, and 0.1% Triton X-100. Lysates were centrifuged at 1000 x g for 5 min at 4ºC, and supernatants were collected as cytoplasmic fractionated lysates. Remaining cell pellets were additionally lysed for 15 min on ice in RIPA buffer, sonicated for 10 s, and collected as
nuclear fractionated lysates. Fractionated lysates were clarified by centrifugation at 15,000 x g for 10 min at 4ºC, and then, normalized for total protein content via Bradford assay. Lysates were resuspended in 5x Laemmli loading buffer. If necessary, immunoprecipitations were performed as previously described. Beads from immunoprecipitated cytoplasmic or nuclear lysates were washed three times with cold Buffer A or RIPA buffer, respectively, and resuspended in 2x Laemmli loading buffer.
2.6.4 Laemmli denaturing lysis
Cells were lysed directly in Laemmli lysis buffer (60 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol), supplemented with fresh 20 mM N-ethylmaleimide (NEM).
Lysates were sonicated twice for 10 s, and then, incubated for 10 min on ice. Lysates were sonicated again for 5 s, and then, clarified by centrifugation at 15,000 x g for 15 min at 4ºC. Protein samples were normalized for total protein content via Bradford assay and resuspended in 5x Laemmli loading buffer. If necessary, lysates were diluted by 12-to-15-fold in Tris-buffered saline and Tween 20 (TBS-T) before performing immunoprecipitations as previously described. Beads were washed four times with cold TBS-T and resuspended in 2x Laemmli loading buffer.
2.6.5 Western blot analysis
Protein samples were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), typically using 8% running gels, and then, transferred to PVDF membranes (Millipore).
Membranes were blocked in 5% bovine serum albumin (BSA) in TBS-T for 1hr at room temperature. Then, membranes were incubated with the indicated antibodies overnight at 4ºC.
Subsequently, membranes were washed three times in TBS-T before incubating in secondary
antibody for 1-2 hr at room temperature. Membranes were washed again three times in TBS-T, and then, visualized using ECL detection reagents.
The following primary antibodies were used for immunoblotting: phospho-ATM/ATR substrate (S*Q) (D23H2/D69H5) (Cell Signaling, #9607), PTEN (138G6) (Cell Signaling, #9559), PARP (Cell Signaling, #9542), FLAG M2 (Sigma-Aldrich, F3165), GAPDH (Santa Cruz, sc-25778), phospho-Chk2 (Thr68) (C13C1) (Cell Signaling, #2197), and SUMO-2/3 (18H8) (Cell Signaling, #4971).
2.6.6 Immunofluorescence assay
Cells seeded overnight on glass cover slips were washed with PBS, and then, fixed with 4% paraformaldehyde for 10-15 min. Using a combined permeabilization/block/primary antibody (P/B/P) solution (0.25% saponin, 0.5% BSA, 10% TBS-T, 1:100 primary antibody, antibody diluent), fixed cells were simultaneously permeabilized, blocked, and incubated with primary antibody for 3-4 hr at 37ºC. The following primary antibody was used for immunostaining: PTEN (138G6) (Cell Signaling, #9559). Samples were washed three times with TBS-T and incubated with goat anti-rabbit IgG Alexa-Fluor 594 (1:250) for 1 hr at 37ºC. Samples were washed again three times with TBS-T and briefly incubated with DAPI (1:1000 in PBS). Cover slips were mounted on to microscope slides using Mowiol and stored overnight at 4ºC. Images were collected using the fluorescent light microscope Zeiss AxioImager and analyzed using Zen Pro, ImageJ, and Prism software programs. Experiments were repeated in replicates of three.