Hif 1a

Cells cultured in six-well plates were rinsed three times with phosphate-buffered saline (PBS) and fixed in 4% polyformaldehyde for 15 min. After permeabilization with 0.5% Triton X-100 for 10 min, cells were blocked with 0.1% BSA for 30 min at room temperature. The cells were incubated with the primary antibody (HIF-1a, 1:200, Novus, NB100-105) overnight at 4°C. After rinsing, cells were incubated with Cy3-conjugated goat anti-mouse IgG (H+L) (1:100, Boster, BA1031) for 1 h at room temperature. Following rinsing with PBS, cells were blocked with 0.1% BSA again and incubated with the primary antibody (YAP, 1:200, CST, #14074) and the secondary antibody FITC-conjugated goat anti-rabbit IgG (H+L) (1:100, Boster, BA1105). We counterstained the cells using DAPI (Solarbio, C0065, China) and visualized them under a fluorescence microscope (Olympus, Japan).

Apoptotic cells were detected using the One Step TUNEL Apoptosis Assay Kit (Beyotime, China). After incubating with the TUNEL reagent in the dark for 1 h at 37°C, cells were stained with 4¢,6-diamidino-2-phenylindole (DAPI, Solarbio, China) for 5 min. Apoptotic cells showed red fluorescence (Cy3 labeled). Images of multiple cells from three independent experiments were obtained with a fluorescence inversion microscope (Olympus, Japan). Beyotime, China) and phosphorylase inhibitor (Servicebio, China). Protein concentration was determined using a BCA protein assay kit (Beyotime). Then, cell lysates were separated by SDS-PAGE (Beyotime) and transferred to PVDF membranes (Millipore). Blots were incubated overnight at 4°C with the following primary antibodies: HIF-1a (1:500, Novus, NB100-105), caspase-3 (1:1000, Proteintech, 19677-1-AP), Bcl-2 (1:1000, Abcam, ab196495), Bax (1:1000, CST, #2772), YAP (1:1000, CST, #14074), p-YAP Ser127 (1:1000, CST, #13008), and b-actin (1:1000, CST, #3700). Immunoblotting was detected using the UVP ChemiDoc-It Imaging System and enhanced chemiluminescence detection kit (Affinity; KF003).