Fig. 5

All colorectal-cancer TCGA DLC1 START mutations show impaired inhibition of migration and anchorage-independent growth, and deficient binding to some or all START binding partners, but intact RhoGAP activity. A Migration in transwells of H358 control (V) or DLC1 wild type (WT) and mutants stable clones. Migrated cells were fixed and photographed (upper panel), and the number of migrated cells were quantified using Image J, from a total of four images from each duplicate experiment (bottom panel). Bars represent mean +/−SD. t-test was performed for statistical analysis (***, ^###:p < 0.001) B. Same stable clones as in A were seeded in soft agar and allow to form colonies for approximately 28 days, and those colonies > 0.2 mm were photographed (upper panel) and counted (lower panel). Each dot represents one plate of cells. 3–5 plates of each stable clone were used for the experiment. Bars represent mean +/− SD. t-test was performed for statistical analysis (***:p < 0.001, ^###:p < 0.005) C. Active Rho (GTP-RhoA) in 293H control (V) or DLC1 wild type (WT) and mutants transient transfectants was analyzed by Rhotekin pull down assays, followed by RhoA Western blot (WB).GFP DLC1 and total Rho expression were also checked by WB. D Glutathione Pull-down (PD) in 293 T cells overexpressing GST control or GST Caveolin-1 (GST CAV) and GFP control (V) or GFP DLC1 wild type (WT) and mutants followed by Western blot (WB) using GFP and GST antibodies. Correct expression of GFP fusion proteins was checked in total lysates (bottom panel). E PLCD1 immunoprecipitation (IP) in 293 T cells overexpressing GFP control (V) or GFP DLC1 wild type (WT) or mutants followed by western blot (WB) using the indicated antibodies. The expression of GFP fusion proteins was checked by Western blot in total lysates (bottom panels)