Fig. 1

GJB3 controls ploidy in Y235T cells. A The presented bar graph illustrates the GJB3 mRNA amounts across various human tissues, with detailed information available in the Materials and Methods section. Urothelial cells (UC#1 and UC#2) were isolated from ureters from two separate patients who underwent nephrectomy at Ulm University. The mRNA levels were normalized to GAPDH. n = 3 independent experiments were performed. Error bars represent mean ± SEM. B The representative pictures display the HE, GJB3 and IgG staining in human ureter tissues (U#1 and U#2, respectively). C The representative Western blot result indicates GJB3 protein levels in Y235T cells with shGJB3. α-tubulin is used as a loading control. n = 3 independent experiments were performed. D The bar graphs depict the effectiveness of GJB3 knockdowns at the mRNA level in Y235T cells, with the measurements reference to the GAPDH mRNA level. n = 3 independent experiments were performed. Error bars represent mean ± SEM. E Representative pictures showing metaphase spreads of Y235T cells with shControl and shGJB3#2. Chromosomes are visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Control cells showing 46 chromosomes in most metaphase spreads. Exemplary pictures demonstrating the induction of aneuploidy in Y235T cells subsequent to GJB3 knockdown. The images show a metaphase spread of Y235T-shGJB3#2 cells with 51 chromosomes. F Chromosomes numbers of metaphase spreads from Y235T cells that were knockdown GJB3. n = numbers of (Each counting is indicated within the graph). Results are pooled from three independent sets of experiments. Mean ± SEM values are shown in the dot plot, and significance was determined by using Fisher’s exact test. G Representative pictures showing micronuclei of Y235T cells with shGJB3#1. Cell nuclei are stained with DAPI, and phalloidin Alexa Fluor 488 was used for F-actin visualization. White arrows indicate micronuclei. H Quantitation of cells with micronuclei upon knockdown of GJB3. Results from n = 3 separate series of experiments. The bar graph displays the mean ± SEM values, and the two-tailed Student's t-test was used to assess the significance. I Immunofluorescence results indicate the multinucleation of Y235T shGJB3#1 cell. Cell nuclei is visualized by DAPI, and F-actin is visualized by Alexa Fluor 488. J Quantitation of cells with multinucleation with knockdown of GJB3. Results from n = 3 independent sets of experiments. Mean ± SEM values are shown in the bar graph, and the significance was determined by two-tailed Student’s t-test. K Figures depict of mitotic abnormalities in metaphase and anaphase. DAPI (blue) indicates chromosomes, Cy5 (red) indicates α-tubulin, and Alexa Fluor 488 (green) labeling illustrates γ-tubulin. White arrows are used to indicate chromatid mislocation or multipolar centrosomes. L–M Quantitative evaluation of mitotic abnormalities. Results from n = 3 distinct experiments. The bar graph displays mean ± SEM data, and a two-tailed Student’s t-test was used to assess significance. Scale bars: 200 μm (B main panels) 50 μm (B insets) 20 μm (E, G, I) and 2 μm (K). Images are shot at total magnification of 100x (B main panels), 630x (B insets, E, G, I, K)