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. 2017 Oct 2;214(10):2889–2900. doi: 10.1084/jem.20170354

Figure 4.

Figure 4.

EGF-driven microcircuitry steers a two-pronged pathway toward metastasis. (A) List of selected interacting partners of DIAPH1 shortlisted from BioID. (B) Western blot analysis on SCC12 lysates immunoprecipitated with anti-DIAPH1 and probed for Arpin (top). Input cell lysates were probed for Arpin (middle) and DIAPH1 (bottom). Representative image is from three independent experiments. (C) Western blot on SCC12 control or shDIAPH1 cell lysates immunoprecipitated with anti-Arpin and probed for Arp3 (top). Input cell lysates were probed for Arp3 (middle) and β-actin (bottom). Representative image is from three independent experiments. IP, immunoprecipitation; WB, Western blotting. (D) Analysis of cell displacement upon DIAPH1 knockdown and rescue with concurrent knockdown of both DIAPH1 and Arpin. Representative image is from three independent experiments. (E) Heat map showing expression values of selected genes from microarray data of SCC12 cells transfected with control shRNA or shFSTL1. Expression values displayed in shades of red (high) or blue (low) relative to the individual mean value of the gene in linear scale. (F) Gelatin zymography and quantification of band intensities represented as a histogram (top). **, P < 0.001; error bars represent SD (n = 3). (G) Western blot on control or shFSTL1 SCC cell lysates from EGF-pretreated cells. Lysates were probed for phosphoERK or total ERK. (H) Western blot showing rescue of phosphoERK upon treatment with recombinant FSTL1 (n = 2). (I) Relative transcript abundance of MMP9 in control or shFSTL1 A253 cells treated with EGF. **, P < 0.001. Error bars represent SD (n = 5). (J) Rescue of MMP9 expression upon treatment with recombinant FSTL1. Error bars represent SD (n = 3). (K) A253 cell lysates were immunoprecipitated with IgG or anti-FSTL1 antibody and probed for Wnt7a by Western blot analysis. Representative image is from three independent experiments. (L) Western blot on lysates from shFSTL1 cells transfected with specific siRNA against Wnt7a and cells treated with EGF posttransfection. Lysates were probed for phosphoERK or total ERK (n = 3).