Fig. 2
GJB3 controls spindle orientation and microtubule dynamics. A Exemplary pictures illustrating the disorientation of Y235T-shGJB3#1 cells. The chromosomes are indicated by DAPI, γ-tubulin is visualized by Alexa Fluor 488, and α-tubulin is visualized by Cy5. B Quantitative assessment of the spindle pole displacement factor (SPDF) in Y235T cells. n = 256 (Y235T-shScr), 253 (Y235T-shGJB3#1), 263 (Y235T-shGJB3#2), C, Representative images showing reorientation of UMUC3 cells with ectopic GJB3. The chromosomes are indicated by DAPI, γ-tubulin is visualized by Alexa Fluor 488, and α-tubulin is visualized by Cy5. D Quantitative assessment of the spindle pole displacement factor (SPDF) in UMUC3 cells. n = 155 (UMUC3-EV), and 140 (UMUC3-GJB3). Experiments present combined data from three separate sets of independent experiments. The two-tailed Student’s t-test was used to evaluate significance, and the mean ± SEM data are displayed in the dot plot. To boost the proportion of prometaphase cells, cells were treated to dimerthylenastron for four hours prior to labeling. E Examples of images demonstrating microtubule growth in Y235T cells expressing shGJB3#2. F Rates of mitotic microtubule plus-end assembly in Y235T-shGJB3 cells. n = 60 cells are pooled from three independent sets of experiments. G Example of images demonstrating growth of microtubules in UMUC3-GJB3 cells. H Rates of mitotic microtubule plus-end assembly in UMUC3-GJB3 cells. n = 60 cells are combined from three separate sets of experiments. The two-tailed Student’s t-test was used to evaluate significance, and the mean ± SEM data are displayed in the dot plot. GJB3 interacts with α-tubulin. The deletion of GJB3 in UROtsa cells using the CRISPR-cas9 method was detailed in the main article. I Western blot displaying GJB3 protein levels in UROtsa cells with a control guide RNAs directed against green flourescent protein or two distinct gRNAs targeting GJB3 (gGJB3#1 and GJB3#2). α-tubulin serves as loading control. n = 3 separate experiments were conducted. J Exemplary pictures displaying GJB3 and α-tubulin colocalization in UROtsa cells during metaphase. GJB3 is visualized by Alexa Fluor 488 and α-tubulin is visualized by Cy5. Yellow signal indicates the overlap of GJB3 and α-tubulin. K Quantitation of GJB3 and α-tubulin colocalization in UROtsa cells by Pearson’s correlation coefficient. n = 49 (UROtsa-gControl), 58 (UROtsa-gGJB3#1), 53(UROtsa-gGJB3#2) are pooled from three to four independent experiments. L GJB3 protein level in UMUC3 cells with ectopic GJB3 was detected by Western blot. α-tubulin is used as a loading control. n = 3 independent experiments were performed. M Representative images displaying the colocalization of GJB3 and α-tubulin in UMUC3 cells during metaphase. GJB3 is visualized by Alexa Fluor 488 and α-tubulin is visualized by Cy5. Yellow signal indicates the overlap of GJB3 and α-tubulin. N Quantitation of GJB3 and α-tubulin colocalization in UMUC3 cells by Pearson’s correlation coefficient. n = 105 (UMUC3-EV), and 64 (UMUC3-GJB3) are pooled from three to four independent experiments. Mean ± SEM values are shown in the bar graph, and significance was determined by two-tailed Student’s t-test (M, N). O–P GJB3 bundle microtubule (MT) filament level was detected by Western blot. 5 × 1011 MT/ml and 5–10 μm in length MTs were incubated with increasing 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. (O), Flag-GJB3, indicated by red arrowheads and (P), microtubules, indicated by red arrows, are visualized by specific antibodies. n = 3 independent experiments were performed. Scale bars: 5 μm (A, C) and 1 μm (E, G) and 2 μm (J, M). Images were captured at total magnification of 630x