Mesenchymal stem cells promote metastasis through activation of an ABL-MMP9 signaling axis in lung cancer cells
Fig 5
Depletion of ABL kinases reduces expression and secretion of MMP9.
PC9 cells were transduced with lentiviruses encoding either scramble control shRNA (SCR) or shRNAs specific for ABL1 and ABL2 (AA). Cells were then co-cultured with or without MSCs followed by FACS sorting to separate PC9-SCR or PC9-AA cells from MSCs. (A) RT-PCR analysis of PC9-SCR, PC9-AA, sorted PC9-SCR and sorted PC9-AA cells for MMP9 mRNA expression. Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparison post hoc testing (***p<0.001; *p<0.05). Error bars represent ± SEM (n = 4). (B) Culture supernatants (SN) from PC9-SCR or PC9-AA cells grown alone or with MSCs under co-culture conditions were analyzed by Western blotting for MMP9 protein and total cell lysates were blotted with indicated antibodies. MMP9 protein in supernatants was normalized to total MMP9 in lysates and presented as fold change. (C-D) Culture supernatants of PC9-SCR or PC9-AA with or without MSC co-culture were analyzed for MMP9 (C) and MMP2 (D) using gelatin-zymography. A representative zymographic band is shown (top) with corresponding band quantification (bottom). Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparison post hoc testing (***p<0.001, **p<0.01, ns = not significant). Error bars represent ± SEM (n = 3).