Fig. 2.

CAFs-derived TSP-4 signaling facilitated EMT and cancer stem-like traits in GBC cells. a GBC-SD and NOZ cells were incubated with CM-shVector, CM-shTSP-4, CM-shTSP-4 + rh-TSP-4 for 24 h, then the invasive ability of GBC cells was assessed by the Matrigel-invasion assay. Scale bars = 50 μm. n = three independent experiments, *P < 0.05 or **P < 0.01 by ANOVA. b Representative images of the tumorsphere formation assay after CM-shVector, CM-shTSP-4, CM-shTSP-4 + rh-TSP-4 treatments in GBC-SD and NOZ cells. The number of tumorspheres was counted and plotted, and the percentage of tumorspheres with diameters of 50–100 μm, 100–150 μm or > 150 μm was calculated and plotted. Magnification is × 200, and scale bars = 50 μm. n = three independent experiments, **P < 0.01 by ANOVA. c The expression of CSC and EMT markers after CM-shVector, CM-shTSP-4, CM-shTSP-4 + rh-TSP-4 treatments were evaluated by western blotting. d The ALDH+ cells populations in NOZ cells after CM-shVector, CM-shTSP-4, CM-shTSP-4 + rh-TSP-4 treatments were detected by Flow cytometric analysis. n = three independent experiments, *P < 0.05 or **P < 0.01 by ANOVA