Figure 5. Tensin2 PTB binding was not necessary for DLC1 to suppress the formation of actin stress fibers but was required to suppress colony formation.

(A) SMMC-7721 HCC cells were transfected with the indicated Myc-tagged DLC1 constructs and the actin stress fibers were stained with TRITC-conjugated phalloidin 1 hour after the serum induction. The asterisk (*) marks the DLC1-transfected cells. DLC1 Y442F, S440A, ΔPTB#1 and #2 could suppress actin stress fiber formation as efficiently as DLC1 WT. Scale bar = 10 µm. (B) HEK293T cells were transfected with the indicated FLAG-tagged DLC1 plasmid for 24 hours. After transfection, the cells were serum-starved for 24 hours and stimulated with 5 µM Lysophosphatidic acid for 30 minutes. Cell lysate was then collected and subjected to GST-RBD pull-down for RhoA-GTP. The pull-down samples were subjected to SDS-PAGE analysis and immunodetected with anti-RhoA antibodies. The band intensity of the active RhoA in each lane was measured and the readings were normalized to the vector-transfected cells. The DLC1, tubulin and RhoA in total cell lysate were immunodetected with anti-FLAG, anti-tubulin and anti-RhoA antibodies, respectively. (C) HeLa cells were transfected with the indicated DLC1 constructs and selected with G418 for 2 weeks. The colonies formed were visualized by crystal violet staining. The mean difference in colony formation efficiency between groups was found to be statistically significant (p<0.001; one-way ANOVA test).