(A) Tube formation: conditioned media were collected from DLD-1 cells treated with single or double knockdown of control, SINHCAF, or HIF-2α, and cultured in hypoxia for 24 h. HUVECs were cultured in the presence of recombinant basement membrane and conditioned media for 24 h. Total tube length was measured by Image J macros, and representative images are shown. N = 3. Graphs depict mean + SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (B) Colony formation: HeLa and DLD-1 cells were seeded for colony formation following siRNA transfection. Relative colony number at day 7 for each cell line is shown. N = 3. Graphs depict mean + SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (C) Control or one of two SINHCAF [1/2] siRNA oligonucleotides were transfected into HeLa cells for 24 h prior to trypsinization and equal number of cells plated on day 1. Cells were counted for two subsequent days. Graph depicts total cell number as the average and SD of three independent experiments. **P ≤ 0.01. (D) Control or SINHCAF siRNA oligonucleotides were transfected to HeLa cells for 48 h. Lysed samples were analyzed by immunoblot for expression of apoptotic and autophagy markers. Graphic depicts the percentage of sub-G1 cells present when analyzed by flow cytometry and represents the average and SEM. *P ≤ 0.05. (E) Control or one of two SINHCAF [1/2] siRNA oligonucleotides were transfected into HeLa cells for 48 h prior to cell fixation. Cells were analyzed by flow cytometry for the cell cycle distribution. Graph depicts the percentage of cells present in each stage of the cell cycle, and it is the average and SD of three independent experiments. *P ≤ 0.05, **P ≤ 0.01. (F) Proposed model for SINHCAF function over HIF-2α and its biological functions.