Validation of the RPE1 inducible Cas9 NuMA KO cell line and of the nocodazole/reversine treatment. (A) Western blot (top) and immunofluorescence measurement (bottom) of endogenous NuMA levels in RPE1 cells after DOX-induced NuMA depletion for the indicated times. n = 147 (0 h), 190 (24 h), 141 (48 h), 107 (72 h), and 64 (96 h) cells. (B) Representative immunofluorescence images showing spindle morphology (tubulin), NuMA, and chromosomes (Hoechst) in the indicated conditions. Cells were untreated or treated with nocodazole (664 nM) and reversine (320 nM) for 18-24 h. Scale bar, 5 µm. Control cells assemble a spindle, NuMA KO cells assemble a perturbed spindle, and nocodazole- and reversine-treated cells do not assemble a spindle. (C) Nuclear morphology in immunofluorescence images of DOX-induced Cas9 RPE1 cells not expressing sgRNAs. n = 209 (no DOX control) and 328 (DOX) cells. (D) Representative immunofluorescence images of nuclear shapes in control and NuMA KO cells after DOX-induced NuMA depletion for the indicated times. The green line outlines the nucleus. Scale bar, 10 µm. (E) Nuclear solidity of interphase cells with respect to endogenous NuMA levels after DOX-induced NuMA depletion for 0–96 h. Whiskers represent minimum to maximum. n = 61 (1.2–1.8), 183 (0.8–1.2), 191 (0.4–0.8), and 214 (0.0–0.4) cells. Two-sided Mann–Whitney test: ***, P = 0.0009; *, P = 0.01; ns, nonsignificant. MN, micronuclei; Noc, nocodazole; and Rev, reversine.