Fig. 4

L-plastin Ser5 phosphorylation is important for migration and invasion of breast cancer cells in vitro. a, b L-plastin (red) and Ser5-P-L-plastin (green) immunoblotting of BT-20 (a) and HCC38 cells (b) transduced with shRNA control (shCTRL) and shRNA targeting L-plastin (shLPL). Cells were treated with 0.1 µM PMA for 1 h. β-actin (red) was stained as a loading control. c, d Statistical plots of transwell migration and Matrigel-coated transwell invasion assays. The number of cells which crossed the membrane was assessed after a 24 h incubation period and five fields at 20 × magnification objective were counted for each well. Three independent experiments were performed for each assay. Results are expressed as means ± SEM. Student’s t-test (*p < 0.05, ***p < 0.001, ****p < 0.0001). E) and F) L-plastin (red) and Ser5-P-L-plastin (green) immunoblotting of BT-549 (e) and MDA-MB-231 cells (f) transduced with GFP or GFP-fused L-plastinWT or the phosphorylation variants L-plastin S5E or L-plastin S5A. Cells were treated with 0.1 µM PMA for 1 h. BT-20 cell extract was loaded as a control for endogenous L-plastin expression. g, h Statistical plots of transwell migration and Matrigel-coated transwell invasion assays. The assays were performed as described under (c, d). Results are expressed as means ± SEM of three independent experiments. One way ANOVA followed by Dunnett’s multiple comparison test relative to GFP transduced cells (*p < 0.05, **p < 0.01, ****p < 0.0001)