FIGURE 3.
Decorin suppresses HIF-1α and VEGFA in constitutively-activated HIF-1α MDA-231 cells under normoxic conditions and suppresses critical transcription factors needed for HIF1A and VEGFA expression. A and B, immunoblot analysis of HIF-1α expression in either wild-type or mutant cell lines as indicated. Cells were exposed for 4 h to increasing concentrations of decorin. The lower parts of the blots were stained with Coomassie to demonstrate equal loading. The migration of molecular mass protein standards is shown on the left. C and D, immunolocalization of HIF-1α and VEGFA in either wild-type or mutant cell lines as indicated. The cells were treated for 4 h with decorin, fixed, permeabilized, and reacted with antibodies specific for either HIF-1α or VEGFA. Bar = 10 μm. E, quantification of secreted VEGFA obtained by slot blot analysis of media conditioned for 4 h by either wild-type or mutant cells as designated. F and G, gene expression analysis of critical pathway genes found to be essential for driving the expression of HIF1A and VEGFA after 4 h (F) or 24 h (G) of decorin as indicated. Data are representative of at least two independent trials run in quadruplicate and normalized to the endogenous housekeeping gene ACTB. Data are reported as percentage change ± S.E. as calculated according to the comparative ΔΔCt method (*, p < 0.05; **, p < 0.01; ***, p < 0.001).