KBP regulates chromosome alignment and spindle length in mitotic cells. (A) Western blot against KBP, showing protein levels under KBP siRNA and mCherry-tagged (mCh) KBP transfection conditions from HeLa cells. γ-Tubulin was used as a loading control. Right: KBP knockdown and mCh-KBP overexpression quantification, obtained from quantification of three independent blots, corrected against γ-tubulin and normalized to the control siRNA transfection. **, P = 0.009; ***, P = 0.0002 by unpaired, two-tailed t test comparing each condition to control siRNA. (B) mCh-KBP does not bind MTs in interphase HeLa cells. Yellow boxes denote inset areas. Arrows highlight occasional mCh-KBP puncta that colocalize with α-tubulin. (C) Representative metaphase HeLa cells arrested in MG132 were treated with control or KBP siRNAs or overexpress (OE) mCh-KBP. (D) Chromosome alignment was quantified by determining the FWHM of a Gaussian fit to the distribution of ACA fluorescence along the spindle axis. Left: Graphical representation of FWHM measurement. Middle: FWHM distance values for each cell under the indicated conditions. Dotted line denotes cutoff value for hyperaligned cells (3.3 µm), empirically determined from the control population. ‡, P = 0.0432 by χ2 analysis comparing hyperaligned populations; ****, adjusted P < 0.0001 with 95% confidence interval by one-way ANOVA analysis with Tukey’s multiple comparisons test of full datasets. Right: Correlation plot of mCh-KBP fluorescence intensity versus FWHM alignment values. Dotted line is linear regression showing the data trend. (E) Left: Plot of spindle lengths measured in cells following the indicated treatments. *, adjusted P < 0.05; ****, adjusted P < 0.0001 with 95% confidence interval by one-way ANOVA with Tukey’s multiple comparisons test. Right: Correlation plot of mCh-KBP fluorescence intensity versus spindle lengths. Dotted line is a linear regression showing the data trend. Error bars represent SD. Data in D and E were obtained from three independent experiments with the following cell numbers: control siRNA (96), KBP siRNA (105), and mCh-KBP OE (34).
