FIG. 7.
RIN1 negatively regulates RTK-mediated cell migration. (A) MDA-MB-231 cells transduced with vector (ctr) or an RIN1 expression construct and MDA-MB-231 cells transfected with siRNA (ctr or RIN1) were allowed to migrate toward HGF. Migration values in all panels represent the means of at least 30 independent field counts. Immunoblots for RIN1 and TUBB (normalization control) are shown above the graph. (B) MDA-MB-231 cells transduced with a vector (ctr), wild-type RIN1, or RIN1R94N were allowed to migrate toward HGF-containing medium for 6 h. Immunoblots for RIN1 and TUBB (normalization control) are shown above the graph. RIN1R94N migrates more slowly due to a carboxy-terminal HTM fusion (as in Fig. 3 and 4) that does not alter function (28). (C) MDA-MB-231 cells were seeded in medium with or without TGF-β (5 ng/ml) and allowed to migrate for 6 h toward target chamber medium with or without HGF (20 ng/ml). “*” indicates that the combined effect is statistically greater than the additive effect by two-way analysis of variance. (D) Conditioned medium (CM) from MDA-MB-231 cells treated, or not, with TGF-β was loaded onto a nylon membrane using a dot blot apparatus. Two independent experiments (#1 and #2) are shown. Standard amounts of HGF were loaded as controls (top row). HGF was visualized using anti-HGF. (E) MDA-MB-231 cells transduced with a control vector (ctr) or a RIN1-targeted shRNA construct and resuspended in medium with or without TGF-β (5 ng/ml) were allowed to migrate for 6 h toward HGF-containing medium. Immunoblots for RIN1 and TUBB (normalization control) are shown above the graph. (F) MDA-MB-231 cells transduced with a control vector (ctr) or a RIN1-targeted shRNA construct were allowed to migrate for 6 h before counting. Where indicated, cells were treated with TGF-β (5 ng/ml; 18 h). Where indicated, the target chamber medium contained IL-8 (100 ng/ml), an epithelial cell chemoattractant.