Fig. 5

GJB3 interactics with F-actin and influences invadopodia formation via actin dynamics. A Representative images demonstrating invadopodia formation of RT4 cells with shGJB3#2. B Quantitation of the invadopodia number was performed in RT4 cells. n = 959 (RT4-shScr), n = 718 (RT4-shGJB3#1), n = 481 (RT4-shGJB3#2). Cells are pooled from three independent sets of experiments. C, Representative pictures showing invadopodia formation of UMUC3 cells upon ectopic GJB3 expression. D Quantitation of the invadopodia number was performed in UMUC3 cells. n = 110 (UMUC3-EV), n = 137 (UMUC3-GJB3). Cells are pooled from three independent sets of experiments. F-actin is visualized by Alexa Fluor 488-phalloidin and Cortactin is visualized by Cy5, respectively. Yellow spots displaying cortactin and F-actin colocalization identify the invadopodia structures. The regions indicated by white boxes are magnified in the insets. The invadopodia are marked with white arrows. The dot plot displays the mean ± SEM data, and the two-tailed Student's t-test was used to assess significance. E Representative pictures indicate the gelatin degradation by RT4 cells upon GJB3 knockdown. F Gelatin degradation capacity of the cells was quantified by measuring the degradation area per RT4 cell. n = 2809 (RT4-shScr), n = 2447 (RT4-shGJB3#1), n = 3544 (RT4-shGJB3#2). G Representative pictures indicate the gelatin degradation by UMUC3-GJB3 cells. H Gelatin degradation capacity of the cells was quantified by measuring the degradation area per UMUC3 cell. n = 1048 (UMUC3-EV), and n = 1246 (UMUC3-GJB3) are pooled from three to four independent experiments. The dot plot displays the mean ± SEM data, and the two-tailed Student's t-test was used to assess significance. I The LifeAct–Ruby signal recovery duration in UMUC3 cells with or without GJB3 is depicted in representative images after LifeAct–Ruby signal bleaching. The white arrows indicate the areas of bleaching. J, The quantitation of bleaching recovery experiments with UMUC3 cells. n = 29 (UMUC3-EV), n = 31 (UMUC3-GJB3). Three different groups of separate experiments' cells are combined. The graphs' data points correspond to the mean ± SEM. P values were calculated using the two-tailed Student's t-test at t = 49 s. K Exemplary pictures displaying the colocalization of GJB3 with F-actin in control UROtsa cells. Insets: enlarged image of the areas shown by white box. L, Quantitation of GJB3 and F-actin colocalization in UROtsa by Pearson’s correlation coefficient. n = 161 (UROtsa-gControl), n = 206 (UROtsa-gGJB3#1), n = 276 (UROtsa-gGJB3#2). M Exemplary pictures displaying the GJB3/F-actin colocalization in UMUC3-GJB3 cells. Insets: enlarged image of the areas shown by white box. N Quantitation of GJB3 and F-actin colocalization in UMUC3 cells by Pearson’s correlation coefficient. n = 672 (UMUC3-EV), and n = 831 (UMUC3-GJB3) are combined from 3 separate experiments. The two-tailed Student's t-test was used to establish significance, and the bar graph displays mean ± SEM results. Alexa Fluor 647 illustrates the F-actin, and Alexa Fluor 488 illustrates GJB3. Yellow highlights denote GJB3 and F-actin overlap. Insets: enlarged images of the colocalized areas shown by white boxes. O, P GJB3 binds bundle actin filaments in a dose-dependent manner by Western blot. Actin (2.5 mg/ml) concentrations of GJB3 (relative GJB3 amount is indicated by + or + +). Supernatant (S) and pellet (P) were subjected to 10% SDS-PAGE after high-speed centrifugation at 100,000 g. Red arrowheads indicate the GJB3, and the red arrow indicates actin filaments visualized by western blot with specific antibodies. n = 3 independent experiments were performed. Scale bars: 10 μm (A, C, E, G, K and M main panels), 1 μm (A, C insets), 2 μm (E, G, K and M insets) and 1 μm (I). Images were captured at total magnification of 630 × (A, C, I, K, M) and 400 × (E, G)