![NuMA/p150Glued localization at the equatorial membrane is not due to failure in cell elongation. (A and B) Confocal live-imaging analysis of HeLa Kyoto cells stably coexpressing AcGFP-NuMA (green) and mCherry-H2B (magenta) that are transfected with control siRNA (A) or siRNA against Ect2 (B). The recording was started 26 h post-transfection for control and Ect2 siRNA transfected cells. “0 min” time-point represents metaphase to anaphase transition, and the images were acquired every minute. Yellow arrowheads depict NuMA localization at the equatorial membrane. The scale bar in this and for the following panels represents 10 μm. (C and D) Schematic representation of quantification method used for analyzing equatorial membrane enrichment of AcGFP-NuMA fluorescence intensity and for computing cell length (d [μm]) (C). Quantification of equatorial membrane enrichment of NuMA, along with the cell length (D) as described pictorially in C. Bar graph represents the cell length for cells that are either transfected with control siRNA (blue), or siRNA against Ect2 (green). The line plot graph represents the equatorial NuMA membrane intensity in cells transfected with control siRNA (violet) or Ect2 siRNA (pink). Time (in min) is shown on the x-axis, and NuMA equatorial intensity together with the cell length is plotted on the y-axis. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. n > 10 cells as listed; error bars: SEM. (E and F) IF analysis of HeLa cells that were either control siRNA transfected and treated with Cdk1 inhibitor RO-3306 (E) or transfected with siRNA-against Ect2 and treated with RO-3306 (F) for 5 min. Cells were fixed and costained using anti-NuMA (green) and anti-p150Glued (red) antibodies. Yellow arrowheads depict NuMA localization at the equatorial membrane. More than 30 cells were visually analyzed, and the representative cells are shown here. The experiments were repeated three times. (G) Visual quantification of the cortical NuMA distribution in RO-3306 or in Ect2 siRNA plus RO-3306 treated cells. The stacked blue bar, and brown bar represent the percentage of the cells that show cortical NuMA distribution at the polar cortical region and equatorial and polar cortical region, respectively. ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. n > 30 cells/experiment, from three independent experiments; error bars: SD.](https://cdn.rupress.org/rup/content_public/journal/jcb/221/12/10.1083_jcb.202203127/3/jcb_202203127_fig2.png?Expires=1771104757&Signature=2lzQjtBQ0aNsnr14~5XLd2T17P6aE39hAzVAPkXXauupzcUQkY81jMzhV2-URWTkfQhvTActDr4BhUO7AWM0lf14pOT4Jf0gJt2JA-GqN~VAWtnWgM20IJlDAoFMVtGrTofchik7ip1pOKYYj-8Nr67S5oastuh0E0nFq1X-Mndfnsrjh7optfhn3Tf8aLH7oPK~qoMUbe7tbJVws~Lg3S2l5ZJqrdRNv38tb~oPBzZgzYb4mCHal2lHSMDZeRpgwjeP4V28bJ5oPm5zeSmxtREUmnnePPL62GhycLkDoAmlYX5HRmWn~mDHA1Bz7zi-2TJqa8ieltufL1Ss-cyPgA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
NuMA/p150Glued localization at the equatorial membrane is not due to failure in cell elongation. (A and B) Confocal live-imaging analysis of HeLa Kyoto cells stably coexpressing AcGFP-NuMA (green) and mCherry-H2B (magenta) that are transfected with control siRNA (A) or siRNA against Ect2 (B). The recording was started 26 h post-transfection for control and Ect2 siRNA transfected cells. “0 min” time-point represents metaphase to anaphase transition, and the images were acquired every minute. Yellow arrowheads depict NuMA localization at the equatorial membrane. The scale bar in this and for the following panels represents 10 μm. (C and D) Schematic representation of quantification method used for analyzing equatorial membrane enrichment of AcGFP-NuMA fluorescence intensity and for computing cell length (d [μm]) (C). Quantification of equatorial membrane enrichment of NuMA, along with the cell length (D) as described pictorially in C. Bar graph represents the cell length for cells that are either transfected with control siRNA (blue), or siRNA against Ect2 (green). The line plot graph represents the equatorial NuMA membrane intensity in cells transfected with control siRNA (violet) or Ect2 siRNA (pink). Time (in min) is shown on the x-axis, and NuMA equatorial intensity together with the cell length is plotted on the y-axis. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. n > 10 cells as listed; error bars: SEM. (E and F) IF analysis of HeLa cells that were either control siRNA transfected and treated with Cdk1 inhibitor RO-3306 (E) or transfected with siRNA-against Ect2 and treated with RO-3306 (F) for 5 min. Cells were fixed and costained using anti-NuMA (green) and anti-p150Glued (red) antibodies. Yellow arrowheads depict NuMA localization at the equatorial membrane. More than 30 cells were visually analyzed, and the representative cells are shown here. The experiments were repeated three times. (G) Visual quantification of the cortical NuMA distribution in RO-3306 or in Ect2 siRNA plus RO-3306 treated cells. The stacked blue bar, and brown bar represent the percentage of the cells that show cortical NuMA distribution at the polar cortical region and equatorial and polar cortical region, respectively. ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. n > 30 cells/experiment, from three independent experiments; error bars: SD.
