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. 2018 Sep 11;7(1):33–53. doi: 10.1016/j.jcmgh.2018.08.007

Figure 6.

Figure 6

SASH1 inhibits CRKL-mediated SRC signaling, which is required for the mesenchymal phenotype. (A) SASH1- and/or CRKL-deficient cells that adhered to fibronectin-coated plates for 3 hours (left panel) or for 24 hours (right panel) were subjected to immunoblot analysis. (B) Immunoblot quantification of SRC (Y416) and paxillin (Y118) phosphorylation after 3 hours of adhesion (Mann–Whitney test; n = 4 independent experiments; P = .0286). ∗P ≤ .05. (C) Cells that adhered for 3 hours to fibronectin-coated plates were subjected to immunofluorescence staining (100× objective, scale bar: 10 μm) for Y118 phospho-paxillin (green), F-actin (red), and 4′,6-diamidino-2-phenylindole for nuclei (blue). (D) The same analysis was performed after 24-hour adhesion to fibronectin-coated plates. (E) Immunoblot analysis of parental HCT116 cells recombinantly expressing RFP as control or CRKL with a C-terminal RFP tag. In addition, CRKL-RFP–expressing cells were treated with 100 nmol/L dasatinib for 24 hours. (F) Parental (wild-type [WT]), CRKL-deficient (C2), SASH1-deficient (S1, S2), and compound SASH1/CRKL-deficient (S1C2, S2C2) cells were treated with 100 nmol/L dasatinib for 72 hours before they were subjected to immunoblot analysis. Mr (K), molecular range (kilodalton); pPaxillin, phospho-Paxillin; pSrc, phospho-Src; RFP, red fluorescent protein.