Figure 1.
NuMA plays a spindle-independent role in the formation of a single, round nucleus. (A) Experimental design for inducible CRISPR/Cas9 NuMA KO RPE1 cells to undergo and exit a spindle-less mitosis. Endogenous NuMA is depleted by DOX addition starting at 0 h, and cells treated with nocodazole (664 nM) and reversine (320 nM) for a spindle-less mitosis starting at 72 h, and subsequently fixed and stained for immunofluorescence at 96 h. (B) Representative immunofluorescence images of nuclear morphologies observed in uninduced NuMA KO RPE1 (control) cells, control cells treated with nocodazole and reversine (for 24 h), 96 h NuMA KO cells, and 96 h NuMA KO cells treated with nocodazole and reversine (for last 24 h). Cells were stained for tubulin (not shown), DNA (Hoechst), and NuMA. Scale bar, 10 µm. (C) Percentage of cells with different nuclear morphologies in the indicated conditions, from DNA images in B. Plot shows mean ± SD n = 1,385 (control), 1,123 (control +Noc+Rev), 638 (96 h NuMA KO), and 649 (96 h NuMA KO +Noc+Rev) cells, from three independent experiments. Two-sided Fisher’s exact test: ****, P < 0.0001; **, P = 0.003; *, P = 0.01. The same nuclear morphologies are compared within different conditions. (D) The solidity of nuclei corresponds to the ratio of the nucleus’ area to its convex hull area. (E) Nuclear solidity in nocodazole- and reversine-treated control and 96 h NuMA KO cells, and corresponding percentage of cells with nuclear solidity 2 SDs below the control mean. Plot shows mean ± SD n = 750 (control) and 656 (96 h NuMA KO) cells, from three independent experiments. Left plot, two-sided Mann–Whitney test: **, P = 0.001. Right plot, two-sided Fisher’s exact test: ***, P = 0.0001. (F) Percentage of cells with different nuclear morphologies in the indicated conditions, from DNA images. Plot shows mean ± SD n = 285 (control), 456 (control +Noc+Rev), 321 (72 h NuMA KO), and 347 (96h NuMA KO +Noc+Rev) cells, from two independent experiments. Two-sided Fisher’s exact test: ***, P = 0.0001; **, P = 0.009; *, P = 0.03. The same nuclear morphologies are compared within different conditions. Fragm., fragmented; MN, micronuclei; Noc, nocodazole; and Rev, reversine.

NuMA plays a spindle-independent role in the formation of a single, round nucleus. (A) Experimental design for inducible CRISPR/Cas9 NuMA KO RPE1 cells to undergo and exit a spindle-less mitosis. Endogenous NuMA is depleted by DOX addition starting at 0 h, and cells treated with nocodazole (664 nM) and reversine (320 nM) for a spindle-less mitosis starting at 72 h, and subsequently fixed and stained for immunofluorescence at 96 h. (B) Representative immunofluorescence images of nuclear morphologies observed in uninduced NuMA KO RPE1 (control) cells, control cells treated with nocodazole and reversine (for 24 h), 96 h NuMA KO cells, and 96 h NuMA KO cells treated with nocodazole and reversine (for last 24 h). Cells were stained for tubulin (not shown), DNA (Hoechst), and NuMA. Scale bar, 10 µm. (C) Percentage of cells with different nuclear morphologies in the indicated conditions, from DNA images in B. Plot shows mean ± SD n = 1,385 (control), 1,123 (control +Noc+Rev), 638 (96 h NuMA KO), and 649 (96 h NuMA KO +Noc+Rev) cells, from three independent experiments. Two-sided Fisher’s exact test: ****, P < 0.0001; **, P = 0.003; *, P = 0.01. The same nuclear morphologies are compared within different conditions. (D) The solidity of nuclei corresponds to the ratio of the nucleus’ area to its convex hull area. (E) Nuclear solidity in nocodazole- and reversine-treated control and 96 h NuMA KO cells, and corresponding percentage of cells with nuclear solidity 2 SDs below the control mean. Plot shows mean ± SD n = 750 (control) and 656 (96 h NuMA KO) cells, from three independent experiments. Left plot, two-sided Mann–Whitney test: **, P = 0.001. Right plot, two-sided Fisher’s exact test: ***, P = 0.0001. (F) Percentage of cells with different nuclear morphologies in the indicated conditions, from DNA images. Plot shows mean ± SD n = 285 (control), 456 (control +Noc+Rev), 321 (72 h NuMA KO), and 347 (96h NuMA KO +Noc+Rev) cells, from two independent experiments. Two-sided Fisher’s exact test: ***, P = 0.0001; **, P = 0.009; *, P = 0.03. The same nuclear morphologies are compared within different conditions. Fragm., fragmented; MN, micronuclei; Noc, nocodazole; and Rev, reversine.

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