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Figure 3 | Cell Communication and Signaling

Figure 3

From: HIF-1α activation results in actin cytoskeleton reorganization and modulation of Rac-1 signaling in endothelial cells

Figure 3

Morphological alterations induced by DMOG are HIF-1α dependent. (A) glEND.2 cells were stably transfected with shGFP, shHIF-1α or shHIF-2α. The clones were cultured under normoxic (N) or hypoxic (H, 1% O2) conditions, or treated with DMOG (D, 1 mM) for 6 h. 30 μg protein of cellular protein were loaded for the detection of HIF-1α, and nuclear extracts (20 μg protein) were used for the detection of HIF-2α. (B) Stably transfected cells, shGFP (GFP), shHIF-1α (HIF1α) or shHIF-2α (HIF2α), were exposed to hypoxia for 20 h. Expression of HIF-target genes phosphoglycerate kinase (PGK) and glucose transporter GLUT-1 mRNA was upregulated in shGFP and shHIF-2α clones but not in shHIF-1α clones. RNA was detected by real time RT-PCR. Expression of PGK or GLUT-1 in GFP cells was set to 1 in each experiment. Data are means ± SD of 4 to 9 experiments. *** p < 0.001, ** p < 0.01 compared to shGFP cells, ANOVA with Tukey’s multiple comparison test. (C) Spheroids obtained form glEND2 cells stably transfected with GFP, shHIF-1α or shHIF-2α were plated on fibronectin and treated with 1 mM DMOG for 24 h. Knockdown of HIF-1α prevented formation of strong F-actin fibers as visualized with PromoFluor phalloidin in shHIF-1α clones. Representative images of shHIF-1α or shHIF-2α cells are depicted. Scale bar: 20 μm. (D) To quantify the areas covered by residual spheroids, cells were stained with Hoechst. Scale bar: 50 μm Areas were quantified in 2 independent experiments with at least 8 spheroids in each group. Data are summarized as means ± SD; mean value of spheroids of control cells was set to 1 in each experiment; error bars of control values represent variability within one experiment. ** p < 0.01; ANOVA with Dunnett’s multiple comparison test.

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