Fig 2: Inactivation of CIP2A disrupts mitotic clustering and disperses pulverized chromosome fragments.a, DOX/IAA-treated mitotic DLD-1 cells with the indicated genotypes with γH2AX-positive Y chromosomes. sg3 and sg4 denote distinct sgRNAs targeting exon 1 of CIP2A. b, Fragment clustering from γH2AX-negative and γH2AX-positive Y chromosomes from a. Data are mean ± s.e.m. Statistical analysis was performed using one-way ANOVA with correction for multiple comparisons compared with the WT controls; ****P ≤ 0.0001. n = 3 independent experiments; from top to bottom, 657, 455, 352, 526, 383, 238, 165, 175, 179 and 149 cells. c, Mitotic CIP2A-KO (sg3) cell with γH2AX-positive Y-chromosome fragments displaced from the genomic mass (arrows). d, Fragment clustering from γH2AX-positive Y chromosomes from CIP2A-KO cells rescued by ectopic CIP2A–HaloTag. Data are mean ± s.e.m. Statistical analysis was performed using two-tailed t-tests compared with CIP2A-KO cells; ****P ≤ 0.0001, ***P = 0.0002. n = 3 independent experiments; 137 (WT), 200 (no rescue), 139 (full length (FL)) and 115 (ΔNES) cells. e, Schematic of inducing the depletion of CIP2A fused to a FKBP12(F36V) degron with dTAGv-1 in mitosis-synchronized cells. noc., nocodazole. f, Immunoblot analysis showing rapid degradation of CIP2A–FKBP12(F36V) after 4 h treatment of mitotic cells with dTAGv-1. Async, asynchronous; sync., synchronized. g, Clustered and dispersed Y-chromosome fragments after mitotic CIP2A depletion. h, Fragment clustering from γH2AX-positive Y chromosomes from g. Data are mean ± s.e.m. Significance was determined using two-tailed Student’s t-tests. n = 3 independent experiments; 194 (mock) and 188 (dTAGv-1) cells. For a,c and g, scale bars, 5 μm.Source Data

Fig 3: Characterization of human DLD-1 cells harbouring biallelic deletions in CIP2A.a) Immunoblot confirmation of CIP2A KO clones. b) Growth curves of WT and CIP2A KO cells with and without DOX/IAA treatment over the indicated number of days. Data represent mean ± SEM; n = 3 biological replicates. c) Flow cytometry analysis of propidium iodide-stained WT and CIP2A KO cells showing similar cell cycle distribution profiles. d) Proportion of micronucleated cells with or without 2d DOX/IAA induction, as determined by DAPI staining. Data represent mean ± SEM; WT: **P = 0.0017, sg3: **P = 0.0023, sg4: *P = 0.0261 by two-tailed t-test compared to untreated controls; n = 3 independent experiments; WT (-DOX/IAA = 5,521, +DOX/IAA = 3,718), CIP2A KO sg3 (-DOX/IAA = 3,436, +DOX/IAA = 2,450), CIP2A KO sg4 (-DOX/IAA = 3,999, +DOX/IAA = 2,930 cells). e) Frequency of Y chromosome fragmentation among Y chromosome-positive metaphase spreads following 3d DOX/IAA induction. Data represent mean ± SEM; not significant (ns), P > 0.05 by two-tailed t-test compared to WT controls; n = 3 independent experiments; WT = 234, CIP2A KO sg3 = 269, CIP2A KO sg4 = 284 cells. f) Mitotic CIP2A KO cells exhibiting fragment dispersion with and without cell cycle arrest with the indicated mitotic inhibitors. Dispersion frequencies and the number of cells analysed are shown. g) Immunoblot of ectopic CIP2A-HaloTag complementation in CIP2A KO cells generated by a frameshift deletion in exon 3 induced by Cas9 ribonucleoprotein (sgRNP) delivery. FL, full length; NES, nuclear export signal. Source Data

Fig 4: Assessment of the effect of CIP2A knockdown on rotenone induced PD pathology. a, c Western blotting assays were used to evaluate the interference effects of the three siRNAs on the CIP2A expression. b, d IF staining of CIP2A (green) confirmed the effect of CIP2A knockdown by siCIP2A1. e, f, g, h Representative IF staining of the p-α-syn and α-syn in the control, siRNA-treated, rotenone-treated, or siRNA + rotenone-treated group. i The PP2A activity was decreased after rotenone exposure but increased after siCIP2A treatment. k, j The α-syn expression decreased after treatment with the three pairs of CIP2A siRNAs. l, n CIP2A interacts directly with α-syn. Co-IP and western blot analysis of the CIP2A and α-syn from the SH-SY5Y cells; normal rabbit IgG served as the controls. m Confocal fluorescence microscopy showing colocalization of CIP2A and α-syn. Values are given as means ± SEM. Statistical significance was determined by one-way ANOVA. Histograms summarize the mean IF signal intensity detected ± SEM as measured in gray values. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 5: Evaluation of CIP2A expression in MPTP induced subacute mouse models. a, l Representative IF images and the quantification of the number of TH + neurons in the SN at 14 days after treatment with MPTP, n = 4 to 6 brains per group. b Behavioral test of the mice showed decreased time in rotarod test and extended time in pole test after MPTP treatment. Data were expressed as means ± SEM, n = 8 to 9 mice per group. c Compared with the control group, there was suppression of the PP2A activity of the brain tissue of the MPTP-treated mice, n = 6 brains per group. d, o Immunoblot results demonstrating the decreased TH expression in both the SN and striatum of the MPTP-treated mice; β-actin served as the loading control. e, p Immunoblot results demonstrating CIP2A expression in the SN of the mice. f Immunoblotting of α-syn. g, h, m, n IF staining of CIP2A (green) and TH (red) as well as IHC assay showed increased CIP2A expression in the SN after MPTP treatment, n = 4 to 6 brains per group. i Plasma CIP2A levels were significantly decreased in the MPTP treated mice when compared to those in the controls. j Immunoblotting of p-α-syn in the SN of rotenone model. k IHC staining of TH and CIP2A in the SN of rotenone model. Histograms summarize the mean IF signal intensity detected ± SEM as measured in gray values. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001