Anti caspase 9

Western blotting was conducted as described in a previous study (30 (link)). The cells were harvested and lysated. The protein concentration was measured using a bicinchoninic acid assay kit (Thermo Fisher Scientific, IL, USA). The protein samples were run on sodium dodecyl sulphate (SDS)-polyacrylamide mini-gels (Bio-rad Mini Protean III) and then transferred onto nitrocellulose membranes by electroelution. Antibodies used in this study included anti-HIF 1α (#610958), anti-caspase 9 (#551246) (BD Bioscience, NJ, USA), anti-ERK 1/2 (#4696), anti-phospho-ERK 1/2 (#9101), anti-AKT (#9272), anti-phospho-AKT (Ser473) (#9271), anti-phospho-mTOR (#5536) (Cell Signaling Technology, MA, USA), anti-mTOR (Thermo Scientific Pierce, PA1-518, IL, USA), anti-RAPTOR (Abcam ab5454, CA, USA), anti-actin (sc-1616), anti-NOX 4 (sc-20141), anti-p22phox (sc-20781), anti-p47phox (sc-14015), and anti-p67phox antibody (sc-15342) (Santa Cruz, CA, USA). Incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, CA, USA) was followed by band visualization using an enhanced chemiluminescence substrate (Thermo Fisher Scientific, IL, USA). The density of the bands was quantified by the GS-700 Imaging Densitometry (Bio-rad, CA, USA), and their values were normalized to that of the actin protein in the control.

The paraffin wax sections were washed with PBS after antigen retrieval as described above. The sections were blocked for 30 minutes at RT, and then incubated with anti‐caspase8 (CST), anti‐caspase9 (CST), anti‐γH2AX (BD biosciences) and anti‐p53 (Ruiyingbio) at 4°C overnight. The sections were then incubated with fluorescent secondary antibody at 37°C for 1 hour avoiding light. Finally, DAPI was added, and the sections were sealed. Laser confocal photographs were taken followed by statistical analysis. Five sections of small intestinal tissue were taken for staining in each group, and three fields of view were selected for counting in each section.