SINHCAF is a repressor of HIF-2α protein in multiple cell lines.
(A) Control or one of the two SINHCAF [1/2] siRNA oligonucleotides were transfected into A549 and HeLa cells cultured in the presence of hypoxia for 24 h. Lysed samples were analyzed by immunoblot for expression of HIF system isoforms and SINHCAF. (B) Control or SIN3A siRNA oligonucleotides were transfected to A549 and HeLa cells cultured in normoxia or hypoxia for 24 h. Lysed samples were analyzed by immunoblot for expression of HIF system isoforms and SIN3A. (C) Expression of HIF-2α following knockdown of SINHCAF and exposure to hypoxia for 24 h was determined in breast MDA-MB-231 and two colorectal (SW480, DLD-1) cancer cell lines. (D) SINHCAF was overexpressed in HeLa and MDA-MB-231 cells with or without exposure to hypoxia for 24 h. Lysed samples were analyzed by immunoblot for expression of HIF system isoforms and SINHCAF. (E) Control, SINHCAF, and PHD2 were singly or doubly knocked down in HeLa cells and expression of the HIF system isoforms was determined by immunoblot. (F) Control and SINHCAF siRNA oligonucleotides were transfected into HeLa cells. Where indicated, cells were starved or serum for 24 h, or serum-starved and serum-added for the final 6 h prior to harvest. MG132 was added for the final 6 h in all conditions. Representative images from at least three experiments are shown.