Micronuclear envelope rupture incidence does not strongly correlate with initial chromatin area, micronuclear area, centromere presence, or γH2AX DNA damage status. (A) Plot of initial chromatin area, a proxy for chromosome size, for micronuclei forming in KIF18A KO or RPE1 nocodazole washout–treated cells. n = 35 (RPE1 + nocodazole), n = 19 (KIF18A KO untreated), n = 31 (KIF18A KO + nocodazole), n = 16 (KIF18A KO + DMSO). P = 0.73, ns. Data points indicate individual micronuclei. (B) Plot showing area of DAPI-stained micronuclear chromatin in an asynchronous, fixed-cell population parsed by completeness of lamin A/C signal for KIF18A KO or RPE1 nocodazole washout–treated cells. n = 219 (RPE1 + nocodazole), n = 310 (KIF18A KO). KIF18A KO intact versus ruptured micronuclei, P = 0.06, ns.; RPE1 + nocodazole intact versus ruptured micronuclei, P = 0.73, ns. (C) Representative images showing DAPI (DNA, to indicate micronuclei) along with antibodies against lamin A/C (to assess micronuclear envelope integrity), ACA (to assess centromere presence), and γH2AX (to assess DNA damage) associated with micronuclei arising in KIF18A KO cells and RPE1 nocodazole washout–treated cells. (D) Plot showing micronuclei positive for ACA signal (top graph; ACA signal indicates that micronuclei likely contain whole chromosomes, while loss of centromeric signal suggests fragmentation) and γH2AX (bottom graph; γH2AX indicates foci of DNA damage) by method of micronuclear induction and p53 status. Data are pooled from three independent experiments. n = 262 (RPE1 control KD), n = 304 (RPE1 control + p53 KD), n = 398 (KIF18A KO control KD), n = 359 (KIF18A KO control KD + p53 KD), n = 297 (RPE1 MAD2 KD), n = 295 (MAD2 KD + p53 KD), n = 312 (RPE1 + nocodazole washout), n = 518 (RPE1 1 Gy, control KD), n = 620 (RPE1 1 Gy, control KD + p53 KD). *, P < 0.0001 (ACA). Indicated P values for numerical data were obtained using unpaired Student’s t test for comparisons between two conditions or one-way ANOVA with Tukey’s post hoc test for comparisons among more than two conditions. Indicated P values for categorical data were calculated using χ2 analysis. Noc., nocodazole.
