Fig. 4

WISP-1 acts through integrin αvβ3 in 344SQ cells and αvβ5 in CAFs to inhibit TGF-β1-induced migration and invasion. a Phase contrast microscopy (left) and quantification of migrated and invaded 344SQ cells (right). 344SQ cells were pretreated with an anti-integrin blocking antibody (3 μg/ml; anti-integrin αν, α5, β1, β3, or β5) or corresponding IgG isotype control for 30 min before treatment with rWISP-1 (10 ng/ml) and TGF-β1 (10 ng/ml) for 24 h. b Phase contrast microscopy (left) and quantification of migrated and invaded CAFs (right). CAFs were pretreated with an anti-integrin blocking antibody (10 μg/ml) for 1 h before treatment with rWISP-1 (3 ng/ml) and TGF-β1 (10 ng/ml) for 24 h. Scale bars: 100 μm. Immunoblot analysis of the indicated proteins in 344SQ cell (c, e) and CAF lysates (f, h). qRT‒PCR analysis of MM2 and MMP12 mRNA expression in 344SQ cell (d) and CAF (g) samples. c–h 344SQ cells and CAFs were pretreated with an anti-integrin blocking antibody (anti-integrin αν, β3, or β5) before direct exposure to rWISP-1 (10 ng/ml for 344SQ cells, 3 ng/ml for CAFs) with or without TGF-β1 (10 ng/ml) for the indicated time. NS: not significant, **P < 0.01, ***P < 0.001, two-tailed Student’s t test. The data are from one experiment representative of three independent experiments with similar results (a, b left, c, e, f, h) or from three independent experiments (mean ± standard error; a, b right, d, g)