Hypoxia-Induced Invadopodia Formation Involves Activation of NHE-1 by the p90 Ribosomal S6 Kinase (p90RSK)
Figure 3
Hypoxia-induced NHE-1 activity promotes invadopodia formation.
HT-1080 cells exposed to normoxic (empty bars) or hypoxic (filled bars) conditions were cultured on a layer of Oregon Green-labeled gelatin for 10 h and stained with Texas Red-phalloidin (actin) and DAPI (nucleus). The percentage of cells that formed invadopodia was determined by fluorescence microscopy. (A) Percentage of invadopodia-forming cells cultured in medium at pH 7.4 or 6.6 (n = 3). (B) Representative confocal microscopy image showing actin staining at sites of Oregon Green-gelatin degradation. Boxed area is enlarged in the corresponding lower panel. The merged image also shows a reconstruction of the x-z profile (inset) through the plane indicated by the solid line within the image. (C–D) Percentage of invadopodia-forming HT-1080 cells (C) transfected with pNHE-1 or an empty vector (pECE) (n = 3) or, (D) transfected with control (scrambled) or NHE-1 shRNA (n = 3–4). Number of invadopodia formed per cell (actin/cortactin clusters), and corresponding area of degradation per cell, quantified from 20 random fields per experiment, in cells (E–F) transfected with pNHE-1 or an empty control vector (pECE) (n = 2–3) or, (G–H) transfected with NHE-1 shRNA or control (scrambled) shRNA (n = 2–3). Columns represent the mean ± SEM indicated by the horizontal bars. The asterisks correspond to, * p<0.01; ** p<0.001; *** p<0.0001.