Following the aforementioned treatments, the cells were harvested and homogenized in RIPA buffer at 4°C for 30 min. The protein concentration was determined by the BCA assay (Beyotime Institute of Biotechnology). Protein (30 µg) from each sample was resolved by 10% SDS-PAGE, followed by transfer to a PVDF membrane (Bio-Rad Laboratories, Inc.). Following blocking with 5% non-fat dry milk at 37°C for 30 min, the membranes were probed with anti-HIF-α (1:1,000), anti-VEGF (1:1,000), anti-P27 (1:1,000), anti-CDK4 (1:1,000), anti-cleaved caspase-9 (1:1,000), anti-cleaved caspase 3 (1:1,000), anti-Akt (1:1,000), anti-P-Akt (1:1,000), anti-P38 (1:1,000), anti-P-P38 (1:1,000), anti-p44/42 MAPK (1:1,000), anti-P-p44/42 MAPK (1:1,000), anti-ARA1 (1:200), anti-ARA3 (1:200) and anti-GAPDH (1:1,000) primary antibodies at 4°C overnight. Subsequently, the membranes were washed with PBS + 1% Tween-20 three times and incubated with horseradish peroxidase-conjugated anti-rabbit or mouse IgG (1:5,000) at 37°C for 1 h. The signals were visualized using an ECL Plus western blot detection system. The grayscale values of the bands were calculated using Image J software (version 1.42) and presented as the fold-change relative to the control group.

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