Fig. 5

HGF-induced expression and secretion of MMPs requires ILEI, efficient ILEI secretion requires c-MET signaling. a Western blot analysis of ILEI secretion and expression, and c-MET and Erk activity and expression in the five selected cell lines (parental) and their control (shCont) and ILEI KD (sh261 and sh506) derivatives after crizotinib (500 nM) treatment for 24 h. b qPCR analysis of MMP-9 (for NCI-H441, MKN45 and OE33) and MMP-2 (for NCI-H1993 and SKBR3) mRNA expression in control (shCont) and ILEI KD (shILEI) cells after 24 h of HGF treatment (40 ng/ml) in the absence or presence of crizotinib (500 nM). Data are normalized as fold change to untreated control cells. Error bars represent SEM of three independent experiments. Statistical significance was determined by one-way ANOVA. c Secretion of MMP-9 and MMP-2 by control (shCont) and ILEI KD (shILEI) NCI-H441 and NCI-H1993 cells treated with HGF (40 ng/ml) for 24 h in the absence or presence of crizotinib (500 nM) determined by gelatin zymography from harvested conditioned medium. The three lanes of each treatment group represent samples of three independent assays. Recombinant pro-MMP-9 was used as assay control. d Quantification of the gelatin zymography gels shown in C. Relative differences in secreted MMP-9 and MMP-2 levels were determined by ImageJ analysis and normalized to HGF treatment-induced control cells. Error bars represent SEM of three independent experiments. Statistical significance was determined by Student’s t-test. e qPCR analysis of E-cadherin mRNA expression (CDH1) in NCI-H1993, NCI-H441, MKN45, OE33 and SKBR3 control (shCont) and ILEI KD (shILEI) cells treated or non-treated with crizotinib (500 nM) for 24 h. Data are normalized as fold change to untreated control cells. Error bars represent SEM of three independent experiments. Statistical significance was determined by one-way ANOVA and marked with asterisks (*p < 0.05; **p < 0.01). f Representative Western blot analysis of E-cadherin expression in the control (shCont) and ILEI KD (shILEI) derivatives of the five selected cell lines after crizotinib (500 nM) treatment for 24 h