Fig. 7: MT1-MMP from the conditioned medium interacts with recipient cells important for gelatin degradation.
From: Intercellular transfer of cancer cell invasiveness via endosome-mediated protease shedding
a Oregon GreenTM 488 labelled gelatin was fixed in 3% formaldehyde before seeding of HeLa cells. After 4 h, the cells were fixed or not before addition of CM from MDA-MB-231-TKS5-GFP-mCh-MT1-MMP cells for 24 h. Graph represents the relative amount of the area of gelatin degradation per cell, mean +/− SD from n = 3 independent experiments. Live, 326 cells, Fixed, 333 cells, one-sample two-sided t-test. b HeLa cells on Oregon GreenTM 488 gelatin treated with CM for 24 h. Areas of degraded gelatin (dark) partly overlap with actin (Alexa568Phalloidin). Representative of 12 images from two independent experiments. c Detection of mCh-MT1-MMP on the surface of HeLa cells treated with CM for 2 h. Representative of 18 images from two independent experiments. d HeLa cells seeded on fibronectin, treated with CM from MDA-MB-231-TKS5-GFP-mCh-MT1-MMP or culture medium for 2 h and stained with anti-mCh and anti-Zyxin (focal adhesion marker). Representative of 22 images from three independent experiments. e Detection of mCh-MT1-MMP in endosomes of HeLa cells treated with CM for 2 h. Representative of 50 images from three independent experiments. f WB of CM from MDA-MB-231-TKS5-GFP-mCh-MT1-MMP cells (left lane) and WCL from HeLa cells treated or not with CM for 4 h. mCh-MT1-MMP full length and the 50–52 kDa ectodomain can be detected in lysate from the CM-treated HeLa cells. * MT1-MMP variants likely comprising processed forms of MT1-MMP and mCh-MT1-MMP. Representative of three WB. g HeLa cells depleted or not for clathrin heavy chain (HC) were seeded on Oregon GreenTM 488 gelatin and treated with CM for 24 h. Graph represents the relative area of gelatin degradation per cell, mean +/− SD from n = 3 independent experiments. In total >290 cells were analysed per condition. One sample two-sided t-test. h HeLa cells seeded on Oregon GreenTM 488 gelatin were incubated with isotonic or hypertonic (addition of 100 mM NaCl) CM for 24 h. Graph represents the relative area of gelatin degradation per cell, mean +/− SD from n = 3 independent experiments. In total >300 cells were analysed per condition. One sample two sided t-test.
