Fig. 4
FAK-Y861F pericytes induce apoptosis in LLC tumour cells. a R&D proteome profiler array using lysates from FAK-WT, FAK-Y397F and FAK-Y861F pericytes. Representative dots of differentially expressed proteins are given. Bar charts show mean fold change in protein expression relative to levels in WT. N = 2 dots from 1 experiment. b Lewis lung carcinoma (LLC) cells incubated with conditioned medium (CM) from pericytes plus or minus recombinant MCP-1. Treatment with FAK-Y861F pericyte CM increased LLC apoptosis, compared with CM from FAK-WT pericytes. This effect was rescued upon treatment with MCP-1 (10 and 50 ng/ml). Bar chart represents % TUNEL-positive cells + s.e.m. Representative images show effect of CM and MCP-1 on LLC cells. Arrows, TUNEL-positive cells. *P < 0.05. One-way ANOVA. N = 5–11 fields of view/genotype. Scale bar, 500 μm. c Early and late stage tumours from PdgfrβCre + ;FAKY861F/Y861F mice had significantly larger TUNEL-positive areas than tumours from PdgfrβCre + ;FAKWT/WT and PdgfrβCre + ;FAKY397F/Y397F mice. Bar chart shows % TUNEL-positive area/tumour + s.e.m. **P = 0.0012, *P = 0.0464; ns, not significant; n = 6 PdgfrβCre + ;FAKWT/WT mice, 8 PdgfrβCre + ;FAKY397F/Y397F mice and 6 PdgfrβCre + ;FAKY861F/Y861F mice. Two-way ANOVA. Representative images show TUNEL-positive staining in tumours. Scale bar, 2.5 mm. d LLC cells incubated with CM from pericytes treated with Src siRNA, non-specific scrambled siRNA (Scr) or transfection reagent (NT). CM from non-treated (NT) Y861F had a significantly higher effect on LLC apoptosis compared with CM from either WT or Y397F pericytes. Knockdown of Src in both WT and Y397F pericytes significantly increased LLC apoptosis. Src knockdown in Y861F pericytes did not increase LLC apoptosis above control levels. Bar chart represents % TUNEL-positive cells ± s.e.m. ***P = 0.0006, *P = 0.0196 One -way ANOVA. ***P = 0.0006 WT NT vs. 861F NT, *P = 0.03 (Y397F NT vs. Y861F NT). Two-sided students t test. N = 10–23 fields of view. Scale bar, 500 μm; arrows in b and d indicate TUNEL-positive cells