Fig. 2

VCE-004.8 activates HIF-1α through a PP2A/B55α pathway. A HEK-293T cells were transfected with B55α or scrambled (siControl) siRNAs, after 2 days in culture treated with VCE-004.8 5 μM for 6 h, and protein expression was analyzed by immunoblotting. B HEK-293T cells were preincubated with the indicated concentrations of either LB-100 or OA for 30 min and then stimulated for 6 h with VCE-004.8. The protein expression was detected by western blot. C, D HMEC-1 cells were preincubated with the indicated concentrations of either LB-100 (C) or OA (D) and stimulated for 3 h with VCE-004.8. The expression of HIF-1α and B55α was detected by western blot. E HMEC-1 cells were transfected with B55α or scrambled (siControl) siRNAs, after 2 days in culture treated with VCE-004.8 5 μM for 3 h, and B55α and HIF-1α protein expression was analyzed by immunoblotting. In all the cases a representative western blot of three independent experiments is shown F NIH3T3-EPO-luc were stimulated with the indicated concentrations of VCE-004.8, LB-100 and OA for 7 h, DFX was used as a positive control for HRE/EPO-luc transactivation, and luciferase activity measured in the cell lysates. Data represent the mean ± SD (n = 3) and significance was determined by Unpaired t test. ***p < 0.0001 VCE-004.8 vs Negative control; ###p < 0.001 VCE-004.8 + LB-100 vs VCE-004.8 treated cells; ###p < 0.0001 VCE-004.8 + OA vs VCE-004.8 treated cells