Hif 1α

Whole cell extracts were prepared using a lysis buffer containing 10 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, 50 mM NaF and 1% Triton X100 supplemented with Complete Protease Inhibitor Cocktail and PhosStop tablets (Roche). After measuring the protein content via Bradford, lysates were subjected to SDS-PAGE and separated proteins transferred to PVDF membranes (Millipore, Massachusetts, USA). For co-IP, protein extracts (IP - 1 mg, input - 100 μg) were incubated with 3 μl STK33 antibody (0.36 μg/μl) and Protein G-Sepharose (GE Healthcare). For western blot analysis 100 μp protein was used. Membranes were blocked with 5% non-fat dry milk in phosphate buffered saline (PBS) containing 0.2% Tween 20 and incubated over night at 4°C with specific antibodies. For subsequent washes 0.2% Tween 20 in PBS was used. The following antibodies were used: STK33 (Abnova, clone 4F7, #H00065975, dilution 1:1000); HIF-1α (BD Transduction Laboratories, #610959, dilution 1:50); cleaved PARP (Cell Signaling, #9505S, dilution 1:700); cleaved caspase 3 (Cell Signaling, #9661, dilution 1:500); HSP90β, (#clone D-19, Santa Cruz Biotechnology, #sc-1057, dilution 1.500); phospho-PKD/PKCμ - Ser744/Ser748 (Cell Signaling, #2054S, dilution 1:1000) and β-actin (Sigma, #A1978, dilution 1:2000).

All cells were synchronized by 100 nM dexamethasone treatment for 2 h. Then, the medium was replaced with fresh medium. After 24-h incubation, these cells were lysed in Cell Lytic-MT (Sigma). The cell lysates were centrifuged at 15,000 rpm at 4°C for 10 min. The supernatants were stored as whole cell extracts at −80°C until use. For Western blotting, 20- µg protein were resolved on 7.5% sodium dodecyl sulfate polyacrylamide (SDS-PAA) gels and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with Tris-buffered saline (TBS)-Tween containing 5% non-fat dried milk. Proteins were detected using antibodies against HIF1α (dilution, 1: 500; BD Transduction Laboratories, Franklin Lakes, NJ, USA), PER2 (dilution, 1∶1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CRY1 (dilution, 1∶2000; Santa Cruz Biotechnology), CLOCK (dilution, 1∶1000; Thermo Scientific), GAPDH (dilution, 1∶10000; Sigma), and BMAL1 (dilution, 1∶100; mouse monoclonal antibody generated in our lab). We performed four replicate Western blots; a representative blot is shown.

Cells were lysed with RIPA buffer (20 mM HEPES, pH 7.4, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 5 mM sodium fluoride, 10 μg/mL phenylmethylsulphonylfluoride, and 1 mM sodium vanadate) for 15 min at 4°C and centrifuged at 13,000 rpm for 15 min. Lysates were subsequently boiled for 5 min in 5X sample buffer (50 mM Tris, pH 7.4, 4% sodium dodecyl sulfate (SDS), 10% glycerol, 4% 2-thioethanol, and 50 μg/mL Bromophenol blue) at a ratio of 4:1. Protein samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), transferred to PVDF membranes (Millipore, Billerica, MA), and immunoblotted with the following antibodies: HIF-1α (#610958, BD Transduction Laboratories, San Diego, CA, USA), MDH2 (YF-MA14165, Abfrontier, Seoul, Korea), P-ACC (#3661, Cell signaling, Danvers, MA, USA), ACC (#3676, Cell signaling), P-AMPK (#2535, Cell signaling), AMPK (#5831, Cell signaling), and β-actin (#8227, Abcam, Cambridge, UK). Protein expression was visualized on Kodak Biomax X-ray film (Kodak, Rochester, NY, USA).

For protein detection, western blotting and immunofluorescence staining were used. For protein expression analysis, cells were harvested in SDS-Triton lysis buffer. Protein concentration was measured using Bio-Rad DC Protein assay and protein detection using Pierce ECL Western blotting substrate (Thermo Scientific). Antibodies used were: HIF-1α (610959, BD Transduction Laboratories), phospho-H2A.X (Ser139) (2577, Cell Signaling), H2A.X (2595, Cell Signaling), GAPDH (5G4-6C5, HyTest), and β-actin (Ac-74, Sigma-Aldrich).
For immunofluorescence staining, cells were fixed with ice cold methanol. The DNA double strand breaks were visualized using antibody against phosphorylated H2A.X (γH2A.X) (2577, Cell Signaling). The nuclei were stained using Hoechst 33342 (Invitrogen). Cells were imaged with Olympus BX60 (40X). The intensity of the phosphorylated H2A.X staining per cell (minimum 4 optical fields yielding minimum 150 cells per condition) was measured using ImageJ software (NIH, USA). Experiments were done as parallel treatments and each experiment was repeated at least three times.

Cells were lysed in Laemmli buffer, and the protein extract was resolved on 10% or 12% SDS-polyacrylamide gels and transferred to 0.45 μm nitrocellulose membranes. The membranes were then blocked and probed with antibodies against: HIF2α (ab199, Abcam); HIF1α (610959, BD Transduction Laboratories, Franklin Lakes, NJ, USA); β-actin (A3854, Sigma, Saint Louis, MO, USA). Antibody binding was detected by enhanced chemiluminiscence (Clarity, BioRad, Hercules, CA, USA; and SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific, Waltham, MA, USA) and visualized on a digital luminescent image analyzer (Image Quant LAS4000 Mini; GE Healthcare, Chicago, IL, USA).