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. 2022 Oct 14;19(12):1373–1391. doi: 10.1038/s41423-022-00930-w

Fig. 3.

Fig. 3

WISP-1 is required for the antimigratory and anti-invasive effects. a Cytokine array analysis of CAF CM, ApoSQ-CAF CM, or ApoSQ CM for 24 h. b Relative image densities of cytokine spots for ApoSQ-CAF CM and ApoSQ CM compared with CAF CM. c Immunoblot analysis of WISP-1 in CAFs transfected with WISP-1 siRNA (upper). Densitometric analysis of the relative WISP-1 abundance (lower). d ELISA of WISP-1 in CM from CAFs transfected with WISP-1 siRNA for 24 h before treatment with or without ApoSQ for 20 h. Quantification of migrated (e) and invaded (f) 344SQ cells. e, f CAFs were transfected with control or WISP-1 siRNA before exposure to ApoSQ for 20 h. CM was added to 344SQ cells with TGF-β1 (10 ng/ml) for 48 h. Quantification of migrated (g) and invaded (h) CAFs. g, h CAFs were transfected with WISP-1 siRNA before exposure to ApoSQ for 20 h, which the medium was then replaced with fresh medium containing TGF-β1 (10 ng/ml) for 24 h. i Phase contrast microscopy (left) and quantification of migrated and invaded 344SQ cells (right). CAF CM and ApoSQ-CAF CM were treated with a neutralizing anti-WISP-1 Ab (10 μg/ml) or IgG isotype control before addition to 344SQ cells with or without TGF-β1 (10 ng/ml) for 48 h. j Immunoblot analysis of WISP-1 in CAFs transfected with control vector (mock) or the WISP-1 plasmid (WISP-1 O/E) for 24 h (upper). Densitometric analysis of the relative WISP-1 protein abundance (lower). k ELISA of WISP-1 in CM from CAFs transfected with empty vector or the WISP-1 plasmid for 24 h before treatment with or without ApoSQ for 20 h. l Phase contrast microscopy (left) and quantification of migrated and invaded 344SQ cells (right). CM from CAFs transfected with empty vector or the WISP-1 plasmid before treatment with or without ApoSQ for 20 h was added to 344SQ cells with TGF-β1 (10 ng/ml) for 48 h. i, l Scale bars: 100 μm. NS, NS: not significant, *P < 0.05, **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 (c and j upper, i and l left) or from three independent experiments (mean ± standard error; c and j lower, d–h, i and l right, k)