Fig 2: Both 17-AAG and PADI3 can suppress CKS1 expression in HCT116 cells. a Expression profile of PADI3, CKS1, Hsp90, CDK1 and p27kip1 were detected using western blot in colon cancer tissues and their corresponding adjacent tissues at the translational level, GAPDH was used to normalize the relative expression of them; b statistical analysis of western blot results in A; c 5 µM 17-AAG was used to treat HCT116 cells for 24 h, and western blot was used to measure the expression level of CKS1, CDK1 and p27kip1; d Statistic analysis of c; e Overexpression of PADI3 was performed in HCT116 cells, and western blot was used to detect the expression level of Hsp90, CKS1, CDK1 and p27kip1, overexpression of RFP as the control group; f statistical analysis of western blot results in e. GAPDH was selected as the internal control, *indicates p < 0.05 for three independent experiments analyzed by Student’s t test

Fig 3: CRBN deficient fibroblast exhibits G2/M cell cycle arrest. (A, B) PI staining and cell cycle distribution analysis of WT and CRBN KO Primary MEFs in P0 and P3. Cells were stained with PI and analyzed for cell cycle distribution using flow cytometry. Representative images of flow cytometry plots are shown. The graph indicates the distribution of each cell cycle phase with different colors, G0/G1(blue), S(gray), and G2/M(green) phases. (C) Western blots analysis using extracts of MEF cells in the early passage were immunoblotted with the anti-Cyclin A, anti-Cyclin B, anti-Cdk1, anti-p-cdc2, and anti–Tubulin antibodies. Tubulin was used to confirm equal protein loading. (D) Relative band intensities as determined by densitometric analysis of the blots in (C). The results shown are representative of five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005; n.s., not significant.

Fig 4: HU and gemcitabine synergize with RP-6306.a-d,f, ZIP synergy scores at various dose combinations of RP-6306 and gemcitabine (a,b,f) or hydroxyurea (HU) (c,d) in FT282-hTERT TP53R175H parental (WT) (a,c), CCNE1-high (b,d) and HCC1569 (f) cells. Score =10 (red color) represents synergy, =-10 (green) represents antagonism. Values were obtained by analyzing mean data from 3 independent biological replicates with SynergyFinder. Growth was monitored with an Incucyte for up to 6 population doublings. e, Growth inhibition relative to DMSO control of FT282-hTERT TP53R175H parental (WT) and CCNE1-high cells after treatment with the indicated dose of hydroxyurea (HU), RP-6306 or the combination of both. Growth was monitored with an Incucyte for up to 6 population doublings. Data are shown as mean ± s.d. (n = 3). g, QIBC quantitation of FT282-hTERT TP53R175H parental (WT) and CCNE1-high cells with pan-?H2AX in response to the indicated RP-6306/HU combinations. Data are shown as mean ± s.d. (n = 3). h–j, m, Replication stress activates phosphorylation of CDK1-T14. (h) Whole cell lysates of the indicated cells and conditions were immunoblotted with CDK1, CDK1-pT14, cyclin B and Tubulin specific antibodies. Quantitation of cyclin B (i), CDK1 (j) and CDK1-pT14 (m). Data are shown as mean ± s.d. (n = 3). k, l, QIBC quantitation of the fold-change of cytoplasmic cyclin B intensity in EdU+ cells following treatment with either HU (k) or gemcitabine (l) at the indicated doses for 48 h. Data are shown as mean ± s.d. (n = 3). For gel source data, see Supplementary Fig. 1. Source data

Fig 5: PKMYT1 inhibition causes unscheduled CDK1 activation and mitotic entry in CCNE1-high cells.a, QIBC analysis of ?H2AX nuclear intensity, EdU incorporation and DNA content (measured with DAPI) in FT282-hTERT TP53R175H cell lines. Representative QIBC (right) and quantification (left) of cells with pan-?H2AX. b, Representative micrograph showing EdU staining, ?H2AX localization and nuclear morphology of FT282 CCNE1-high cells. Representative of n = 3 experiments. c, QIBC quantification of HCC1569 cells with pan-?H2AX as a function of RP-6306 dose. d, Quantification of FT282-hTERT TP53R175H CCNE1-high cells transfected with siRNAs targeting cyclin B1 (siCCNB1) or non-targeting siRNA (siCTRL) with pan-?H2AX as a function of RP-6306 dose. e, QIBC quantification of FT282-hTERT TP53R175H cells of the indicated genotype positive for EdU and cyclin B (CCNB1) pS126 as a function of RP-6306 dose. f, g, RP-6306 induces chromosome pulverization. Representative micrographs of metaphase spreads of FT282 parental (WT) and CCNE1-high cells left untreated or following treatment with either RP-6306 (500 nM) for 24 h (f, left) and quantification of FT282 cells (f, right) and HCC1569 cells (g) with pulverized chromosomes with at least 40 metaphases counted per replicate. h, i, Quantification of the first observed G2 phase (h) and the number of nuclear envelope breakdowns (NEBDs) before the first observed cell division (i) using time-lapse imaging of FT282-hTERT TP53R175H PCNA–chromobody–TagRFP (WT) and CCNE1-high (CCNE1) cells treated with DMSO or RP-6306 (500 nM) for 23 h. QIBC validation is shown in Extended Data Figs. 3a, 4a, e, f, h, i. Data in a, c–i are mean ± s.d. (n = 3).Source data