Fig. 2

MSI2 is required for CAFs to promote NSCLC cell motility. A Schematic illustration of MSI2 depletion mediated by CRISPR-Cas9 system. The oligo sequences of gRNA against MSI2 is shown. B CAFs were transfected with either lentiviral expressing all-in-one MSI2 gRNA and Cas9 (gMSI2) or an empty vector (control, Ctrl). The depletion efficiency of MSI2 was measured by Western blotting (left). β-actin was used as a loading control. Quantitative analysis of MSI2 level, normalized to β-actin and relative to Ctrl CAFs (right). Data are mean ± SD (n = 3); ^**P < 0.01 versus Ctrl CAFs; two-tailed Student’s t-test. C Workflow diagram showing the collection of CM from Ctrl and gMSI2 CAFs, following by Transwell migration and invasion assays of NSCLC cells. D Loss of MSI2 in CAFs inhibits NSCLC NCI-H292 (upper) and NCI-H460 (lower) migration and invasion upon exposure to CM from CAFs. Representative micrographs of migrating/invading cells stained with Hoechst 33342 are shown. Scale bar = 100 μm. Bar graph depicts relative cell migration and invasion. Data are presented as mean ± SD (n = 3); ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus media; ^*P < 0.05, ^***P < 0.001, ^****P < 0.0001 versus Ctrl CAF-CM; one-way ANOVA with Tukey’s multiple comparison test. E Migration and invasion capabilities of NSCLC NCI-H292 (upper) and NCI-H460 (lower) cells upon direct co-culture with either Ctrl CAFs or gMSI2 CAFs (ratio 1:1). Representative micrographs of migrating/invading cells stained with Hoechst 33342 are shown. Scale bar = 100 μm. Migrated/invaded cells were counted and plotted. Data are mean ± SD (n = 3); ^##P < 0.01 ^###P < 0.001 versus NSCLC cells alone; ^**P < 0.01, ^***P < 0.001 versus NSCLC cells with Ctrl CAFs; one-way ANOVA with Tukey’s multiple comparison test. CAFs were GFP labeled, allowing the specific detection of migrated/invaded CAFs (right). Data are mean ± SD (n = 3); ^**P < 0.01, ^***P < 0.001 versus Ctrl CAFs; two-tailed Student’s t-test