Fig. 3

CDK5RAP3 in GNEC cells reduces secretion of VEGFA into the supernatant via suppression of AKT/HIF-1α/VEGFA signaling. a VEGFA levels in four stable ECC10/ECC12 cell supernatants as determined by ELISA. CDK5RAP3 in GNEC cells reduces secretion of VEGFA. b CDK5RAP3 decreases VEGFA protein levels in ECC10 cells as shown by immunofluorescence staining. Scale bar, 50 µm. c VEGFA expression in four stable ECC10/ECC12 cells detected by western blot analysis. CDK5RAP3 expression is inversely correlated with VEGFA protein expression. d mRNA levels of VEGFA in four stable ECC10/ECC12 cells measured by RT-PCR. e p-AKT (S473) and HIF-1α expression in ECC10 cells detected by western blot analysis. Knockdown of CDK5RAP3 increases protein levels of p-AKT (S473) and HIF-1α. Overexpression of CDK5RAP3 mediated opposing effects. f Lenti-scr and Lenti-shC53 transfected ECC10 cells were treated with 20 μM LY294002 (a highly selective inhibitor of AKT). Effects of AKT knockdown confirmed by western blot analysis. Inhibition of AKT decreases protein levels of HIF-1α and VEGFA in ECC10 cells. Quantification of results is presented in g. Mutant CDK5RAP3 reversed the trend in protein levels for p-AKT (S473), HIF-1α and VEGFA induced by CDK5RAP3 knockdown. Changes in CDK5RAP3 expression were confirmed by western blot analysis (h)