Fig. 4

GJB3 inhibits cells invasion and migration. A–B Western blots displaying the GJB3 protein quantity assessments in RT4 and in UMUC3 cells with experimental modifications. The loading control was provided by α-tubulin levels. The Western blots were repeated for three times. C Cell migratory capacity in RT4 cell line with shGJB3#1 was detected by Wound healing/scratch. The exemplary imaged were captured at 24 and 120 h. D Normalized cell free area was used to quantify the impact of GJB3 knockdown on RT4 cells by Wound healing assay. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student's t-test was used to assess significance. E Cell migratory capacity in UMUC3 cell line with ectopic GJB3 was detected by Wound healing/scratch. The representative pictures captured at 12 and 24 h in case of UMUC3 cells. F Quantitation of normalized cell free area of UMUC3 cells with ectopic GJB3 performed by Wound healing assay n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student’s t-test was used to assess significance. G The invasion capacity of RT4 cell line with shGJB3#1 was detected by Boyden chamber. The exemplary images depict cell invasion through the Boyden chamber, stained at 144 h post-seeding. H Quantitation of invasive capacity of RT4 cells expressing the indicated shRNAs targeting GJB3. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student’s t-test was used to assess significance. I The invasion capacity of UMUC3 cell line with ectopic of GJB3 was detected by Boyden chamber. The exemplary images depict cell invasion through the Boyden chamber, stained at 48 h post-seeding. J, Quantitation of invasive capacity of UMUC3 cells with ectopic GJB3 expression. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student's t-test was used to assess significance. K Representative images of hematoxylin/eosin stainings showing the invasion capacity of RT4 cell line with shGJB3#2 by porcine bladder ex vivo organ culture method (the invasive capacity of BC cells in the ex vivo organ culture model was quantified as shown in Fig. S4). The cells were seeded on the surface of the de-epithelized porcine bladder for 21 days. L Quantitation graphs displaying the impact of GJB3 alteration on the invasive capacity of RT4 cells in ex vivo organ culture approach. n = 4 (RT4-shScr), n = 3 (RT4-shGJB3#1), n = 3 (RT4-shGJB3#2) M Representative images of hematoxylin/eosin stainings showing the invasion capacity of UMUC3 cell line with ectopic GJB3 by porcine bladder ex vivo organ culture approach. The cells were seeded on the surface of the de-epithelized porcine bladder for or 14 days. Insets: enlarged images of the areas shown by black boxes. Black arrows indicate the cells that have spread the farthest from the surface. n = 3 (UMUC3-EV), n = 4 (UMUC3-GJB3) independent experiments were performed. Mean ± SEM values are shown in the bar graph, and significance was determined by two-tailed Student’s t-test. Scale bars: 200 μm (C, E, G, I, K main panels, M main panels) and 100 μm (K insets, M insets). Images were captured at total magnification of 50 × (C, E), 100 × (G, I, K main panels, M main panels), and 200 × (K insets, M insets). O-V Morphological changes and actin-enriched protrusions in UMUC3, and RT4 cells with altered GJB3 expression. Cells with actin-enriched protrusions are marked with white arrows. O-P Brightfield microscopy images depict cells exhibiting a transition towards a round morphology (O) of UMUC3 cells with ectopic GJB3 expression. P RT4 cells with GJB3 knockdown demonstrate an elongated shape. Magnifications indicate 100 × or 400 × , respectively. The scale bars refer to 100 μm (left), and 50 µm (right). Q-T Quantification of round or elongated morphology on fixed cells. A cell with elongated or round morphology is identified by the ratio of longest and shortest diameter of the cell from images captured randomly at 630 × magnification using a Zeiss TCS SP5 confocal microscope. Scale bars: 20 μm. The ratio is calculated as the longest diameter of the cell dividing by the shortest diameter of the cell. The ratio is calculated as longest diameter dividing by shortest diameter. A cells with Ratio ≤ 2 is identified as round morphology, while ratio > 2 is elongated morphology. Q, R Immunofluorescence staining photos illustrate the round morphology shifting of (Q) UMUC3 cells with GJB3 overexpression compared to cells transfected with empty vector (EV). R RT4 cells with GJB3 knockdown display a transition towards an elongated morphology compared to cells transfected with shScramble (shScr). Cell nuclei are stained with DAPI, and F-actin is labeled with Phalloidin-AF488. S, T The bar graphs reveals percentages of cells with different morphology in total in each group, shown above in Q and R. The percentage was calculated as number of cells with elongated (or rounded) morphology divide cell numbers in total. The percentage values in different groups are marked above or in the bars. Black bars indicate percentages of cells with elongated morphology, and gray bars indicate the percentage of cells with rounded morphology. The statistical significance is calculated by using chi-square statistic. U, V The graphs show the fraction of cells with actin-enriched protrusions in response to GJB3 alterations (U) in RT4 or (V) in UMUC3 cells. For each picture, the percentage of cells with actin-enriched protrusions is calculated by the number of the cells with actin-enriched protrusions divided by total number of cells. (n) indicates the number of pictures taken in the group