Figure 4

p53 suppresses Toca-1 expression and invadopodia formation in MTLn3 cells. (A) MTLn3 cells were treated with CPT (4 μM) for indicated times and analyzed by IB with Toca-1, p53 and ERK antibodies (ERK served as a loading control). (B) MTLn3 cells with stable silencing of p53 and Toca-1 alone, or in combination, was tested by IB with Toca-1, and p53 antibodies (α-actinin served as a loading control). (C) Representative epiflourescence micrographs for MTLn3 control, sh-p53 and sh-p53/sh-Toca-1 cells plated on TRITC-gelatin (ECM) and stained with phalloidin (F-actin; merge indicates an overlay of both channels). (D) Graph depicts the ECM degradation area relative to total cell area for cell lines described above (mean ± s.e.m.; triplicate samples with 12 fields and >40 cells analyzed; ^*depicts a significant difference based on paired Student’s t test (P <0.05)). (E) Transwell invasion assays for MTLn3 control, sh-p53 and sh-p53/sh-Toca-1 cells. The graph depicts the number of cells per field that had invaded through the ECM to the underside of the filter for each cell line (mean ± s.e.m.; 12 fields were analyzed from triplicate filters for each cell line; ^* and ^** depict significant differences between the indicated groups (P <0.05 and P <0.01, respectively) based on paired Student’s t test; dashed line indicates no significant difference (n.s.) based on paired Student’s t test; results are representative of two independent experiments). CPT, camptothecin; IB, immunoblot; ECM, extracellular matrix; ERK, extracellular signal-regulated kinases; F-actin, filamentous actin; s.e.m., standard error of the mean; Toca-1, transducer of Cdc42-mediated actin assembly; WT, wild-type.