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. 2023 Jan 11;14:151. doi: 10.1038/s41467-023-35881-x

Fig. 5. Interplay between ANXA1, LGN-NuMA, astral microtubules and cortical F-actin during metaphase.

Fig. 5

a Confocal images of representative metaphase MCF-10A cells transfected with si-Control, si-ANXA1#1 or si-ANXA1#2 stained for α-tubulin (grey) and counterstained with DAPI (DNA, magenta). White arrowheads indicate elongated and buckled astral microtubules. Dashed lines outline the cell contour. b Confocal images of representative metaphase MCF-10A cells stably expressing EB3-GFP and transfected with si-Control, si-ANXA1#1 or si-ANXA1#2. Dashed lines outline the cell contour. c Ratio of astral microtubule and EB3-GFP comets length (I) in siRNA-transfected cells: astral microtubules (si-Control: n = 31; si-ANXA1#1: n = 43; si-ANXA1#2: n = 45); EB3-GFP comets (si-Control: n = 30; si-ANXA1#1: n = 34; si-ANXA1#2: n = 34). I1 and l2 of astral microtubules were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, astral microtubules: **P = 0.007 and *P = 0.035; EB3-GFP: ***P = 0.001 and ***P = 0.0009. d Relative fluorescence intensities of astral microtubules (Iastral, rel) in siRNA-transfected cells (si-Control: n = 31; si-ANXA1#1: n = 47; si-ANXA1#2: n = 47). Fluorescence intensities on the spindle (Ispindle) and total cell (Itotal) were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, P = 0.467 and P = 0.676. e Ratio of pole-to-cortex distance (d) in siRNA-transfected cells (si-Control: n = 40; si-ANXA1#1: n = 48; si-ANXA1#2: n = 52). Pole-to-cortex distance d1 and d2 were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, **P = 0.004 and *P = 0.032. f Confocal images of representative metaphase MCF-10A cells transfected with si-Control, si-ANXA1#1 or si-ANXA1#2 stained for F-actin (grey) and counterstained with DAPI (DNA, magenta). g Cortical to cytoplasmic ratio of actin fluorescence intensities (Icortex/Icytoplasm) in siRNA-transfected cells (si-Control: n = 30; si-ANXA1#1: n = 30; si-ANXA1#2 n = 30). Fluorescence intensities in the cytoplasm (Icytoplasm) and total cell (Itotal) were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, ***P = 0.001 and ***P = 0.0003. h Confocal images of representative MCF-10A cells treated with DMSO (Control) or 1 µM Latrunculin A for 30 min, stained for F-actin (grey) and counterstained with DAPI (DNA, magenta). i Confocal images of representative MCF-10A cells treated with DMSO (Control) or 1 µM Latrunculin A for 30 min, stained for ANXA1, LGN, NuMA or p150Glued (grey), and counterstained with DAPI (DNA, magenta). j Average cortical and cytoplasmic fluorescence intensity profiles of ANXA1, LGN, NuMA and p150Glued from metaphase cells (Control: n = 30; Latrunculin A: n = 30). Asterisks indicate the spindle poles. All data are presented as mean ± s.e.m. from 3 independent experiments. n.s. (not significant). arb. units (arbitrary units). All scale bars, 5 µm. Source data are provided as a Source Data file.