Figure 3

Effect of selenium treatment on HIF-1α, HIF-2α, VEGF and cell growth. (A) MSA inhibits both HIF-1α and HIF-2α. RC2 cells expressing HIF-1α and 786–0 cells expressing HIF-2α were treated with and without 10 μM MSA for 24 h; HIF-1α and HIF-2α were detected by western blot. β-actin expression was used as a loading control. (B) MSA down-regulates secreted VEGF in RC2 cells but not in 786–0 cells. Secreted VEGF was measured by ELISA in ccRCC cells. Cells were treated with and without MSA for 24 h and media were used to measure VEGF in RC2 and 786–0 cells and normalized with protein and expressed as pg/mg protein. Experiment was repeated twice with duplicates and P value < 0.05 was considered as significant. (C). HIF-α inhibition by MSA was associated with growth inhibition. Cells were treated with various concentrations MSA (3, 5, 7 and 10 μM) for 24 h. Medium was removed, rinsed and fresh medium was added and allowed to proliferate for 96 h. Cells were fixed and determined the cell survival by SRB assay. Growth inhibition was presented as percent growth inhibition compared to untreated controls. Experiment was repeated twice with 4–5 replicate samples. *p <0.001. (D). VHL transfected RC2 cells which do not express HIF-1α were equally sensitive to MSA like RC2 cells which express HIF-1α . Expression of HIF-1α in RC2 and VHL transfected RC2 VHL cells with and without MSA (upper panel). Cytotoxic effects of MSA in RC2 and RC2 VHL cells (lower panel). Cell survival was determined by SRB assay. *p <0.001.