Extended Data Fig. 6: Premature interphase recruitment of CIP2A-TOPBP1 to ruptured micronuclei.
From: Mitotic clustering of pulverized chromosomes from micronuclei
a) Examples of CIP2A localization patterns in ruptured (γH2AX-positive) micronuclei of DLD-1 cells. Intensity measurements shown in Fig. 3a. Scale bar, 10 μm. b) Examples of CIP2A localization patterns in ruptured micronuclei in DLD-1 cells expressing cGAS-GFP and immunostained for cGAS accumulation (top) or the lack of acetylated H3K9 (bottom) in RPE-1 cells. Scale bar, 10 μm. c) Intensity measurements of distinct CIP2A localization patterns in micronuclei compared to the cytoplasm in the indicated cell lines. Box plot represents interquartile range with min-max; HeLa: none, n = 322, diffuse, n = 48, puncta, n = 30; RPTEC: none, n = 91, diffuse, n = 100, puncta, n = 69; RPE-1: none, n = 317, diffuse, n = 86, puncta, n = 34; RPE-1 H2AX–/–: none, n = 274, diffuse, n = 47, puncta, n = 40 micronuclei pooled from 3-5 independent experiments. d) Frequency of CIP2A localization patterns in ruptured (γH2AX-positive, cGAS-positive, or H3K9ac-negative) micronuclei across a panel of human cell lines. Data represent mean; from left to right, n = 218, 99, 169, 134, 119, 76, 111, 211, 196, 92, 121, and 67 micronuclei pooled from 2 (RPE-1 H2AX–/–) or 3 (all other conditions) independent experiments. e) Examples of TOPBP1 localization patterns in ruptured (γH2AX-positive) micronuclei of DLD-1 cells. Scale bar, 10 μm. f) Examples of TOPBP1 localization patterns in ruptured micronuclei in DLD-1 cells expressing cGAS-GFP and immunostained for cGAS accumulation (top) or the lack of acetylated H3K9 (bottom) in RPE-1 cells. Scale bar, 10 μm. g) Frequency of TOPBP1 localization patterns in ruptured (γH2AX-positive, cGAS-positive, or H3K9ac-negative) micronuclei across a panel of human cell lines. Data represent mean; from left to right, n = 323, 120, 172, 133, 143, 87, 160, 295, 221, 82, 230, and 88 micronuclei pooled from 3 independent experiments. For (d) and (g), DLD-1 cells were treated with DOX/IAA to induce Y chromosome micronuclei, HeLa and RPE-1 cells were treated with CENP-E/Mps1 inhibitors to induce random micronuclei, and RPTECs were transfected with Cas9 ribonucleoproteins targeting the chromosome 3p arm near the centromere to induce chromosome 3p micronuclei.
