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. 2022 Dec 15;34(1):ar1. doi: 10.1091/mbc.E22-08-0306-T

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© 2023 Laguillo-Diego, Kiewisz, et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution 4.0 International Creative Commons CC-BY 4.0 License.

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FIGURE 2: — Characterization of metaphase spindles in MCRS1-silenced cells. (A) Western blot analysis showing the levels of MCRS1 in control and MCRS1-silenced cells. Tubulin levels were used as a loading control. (B) Immunofluorescence staining of siScramble (top panel) and MCRS1-silenced cells (bottom panel). MCRS1 is shown in green, DNA in blue, and tubulin in magenta. MCRS1 staining can be observed as tiny spots at spindle poles in the control cell (arrows). Upon silencing, MCRS1 cannot be detected anymore. Scale bars, 5 μm. (C) Black/white images of control spindle (top, left) and a silenced spindle (bottom, left). The magenta lines show the angles that were measured for the half-spindle (top, right) and for the MTs in the outward position of the spindle (bottom, right). Scale bars, 5 μm. (D) Three-dimensional models of spindle shape in siScramble and siMCRS1 cells were obtained from low-resolution screening by transmission electron microscopy (Supplemental Table S2). The outline of the spindle volume in each serial section is shown in gray and chromosomes in blue. Scale bar, 1 µm. (E) Boxplot showing the quantification of the half-spindle angle obtained from immunofluorescence images. The boxes show the upper and lower quartiles, and the whiskers show the minimum and maximal values excluding outliers; the line inside the box indicates the median. Asterisks show a significant difference according to a linear regression model considering two different experiments with a p value = 4.729 × 10^–8 (siScramble: n = 105; siMCRS1: n = 110). (F) Scatterplot showing the quantification of the half-spindle angle from electron microscopic images. The line shows the mean, and the error bars represent ± SD. Asterisks show significant differences according to a two-tailed Student’s t test with a p value = 0.017 (siScramble: n = 4; siMCRS1: n = 8). (G) Boxplot showing the quantification of the half-spindle angle obtained from immunofluorescence images. Asterisks show a significant difference according to a linear regression model considering two different experiments with a p value = 7.486 × 10^–26 (siScramble: n = 201; siMCRS1: n = 161). (H) Scatterplot showing the quantification of the MT angle obtained from electron microscopic images (two-tailed Student’s t test with a p value = 0.002 (siScramble: n = 8; siMCRS1: n = 16).