Figure 4

CAIX colocalizes with MMP14 at invadopodia and regulates type I collagen degradation. (a) Images of a MDA-MB-231-CAIX-BirA* cell (white dashed line shows boundary) cultured on type I collagen in normoxia, identifying a ROI (yellow box) containing invadopodia (arrows) labeled for Cortactin (cyan), CAIX (red) and MMP14 (green). Profile plots showing colocalization of CAIX and MMP14 at cortactin-positive invadopodia (1 and 2) along the indicated lines (insets) are shown at the right. Scale bar, 10 μm. (b) Images of an MDA-MB-231-CAIX-BirA* cell (white dashed line shows cell boundary) cultured on DQ type I collagen in normoxia, identifying an ROI (yellow box) containing regions of collagen degradation (1 and 2, arrows) that are labeled for DQ collagen (cyan), CAIX (red) and MMP14 (green). Profile plots showing colocalization of the signals along the indicated lines (insets) are shown to the right. Scale bar, 10 μm. (c) Images showing WT MDA-MB-231 cells cultured in hypoxia on fluorescently labeled gelatin (blue, with black areas denoting regions of degradation) and stained for CAIX (green) and markers of invadopodia (red), including cortactin, Tks5 and MMP14 to demonstrate colocalization at mature invadopodia (white arrows; yellow foci). Scale bars, 10 μm (left), 2 μm (right). (d) Quantification of DQ collagen degradation by the indicated MDA-MB-231 cell lines grown in normoxia. ***P<0.001. (e) Quantitation of degradation of DQ collagen by MDA-MB-231 CAIX KO and CAIX rescue cells cultured in normoxia. *P<0.05. Data in (d) and (e) show the mean±s.e.m. of technical replicates (n=5) and are representative of three independent experiments. (f) Dose-dependent inhibition of DQ collagen degradation by CAIX rescue cells treated with U-104. Data show the mean±s.e.m. of technical replicates (n=4) and are representative of two independent experiments. *P<0.05, **P<0.01. (g) Quantification of DQ collagen degradation by WT MDA-MB-231 and CAIX KO cells cultured in hypoxia. Data show the mean±s.e.m. of technical replicates (n=8) and are representative of three independent experiments. **P<0.01. (h and i) Invasion through Matrigel by highly metastatic MDA-MB-231 LM2-4 breast cancer cells cultured in hypoxia and (h) expressing CAIX-targeted (shCAIX) shRNA or (i) treated with U-104. Data show the mean±s.e.m. of three independent experiments. ***P<0.001. Western blots of hypoxia-induced CAIX expression are shown as insets.