Journal: bioRxiv

Article Title: Mitotic CDK4/6 activity sustains spindle checkpoint signalling to prevent mitotic slippage and genomic instability

doi: 10.1101/2025.09.13.675969

Figure Lengend Snippet: a, HCT116 cells were synchronised in prometaphase using thymidine-nocodazole treatment, followed by 9 hours of nocodazole incubation. Cells were treated with Palbo or a CDK1-specific inhibitor, RO-3306 (CDK1i, 10 µM), with or without MG132. Samples were collected at 3 and 7 hours after treatment for flow cytometry and immunoblot analysis. Mitotic exit (decrease in pH3-positive cells) induced by Palbo, but not by CDK1i, was effectively blocked by proteasome inhibition. Data are means ± s.d. (N = 3); two-way ANOVA. Western blot analysis shows retention of highly phosphorylated Cdc27 and CycB1 in MG132-treated cells. b, Cells were synchronised in prometaphase as above and treated with Palbo or vehicle control in the presence or absence of the APC/C inhibitor, proTAME (25 µM). Flow cytometry analysis revealed that proTAME blocked mitotic exit induced by CDK4/6 inhibition, as evidenced by retention of pH3-positive cells with high CycB1 levels. c, HCT116 cells were arrested in mitosis using nocodazole and co-treated with MG132 to prevent mitotic exit. BubR1 and centromere marker CENP-C were visualised by immunofluorescence. BubR1 kinetochore localisation decreased over time upon Palbo treatment, while it remained stable in control conditions. Alisertib (1 µM) served as a positive control for SAC inactivation. Scale bar: 10 µm. Pearson correlation coefficients (PCC) quantify BubR1/CENP-C co-localisation; one-way ANOVA. n = 20, 100, 100, 100 (-); 20, 99, 100, 100 (+Palbo); 21, 100, 98, 100 (+Alisertib), at T0, T1, T2, T3 in a single experient. d, CDK4/6 inhibition reduces KNL1 MELT phosphorylation (pMELT) and BubR1 phosphorylation. Left: Immunoblots show decreased pMELT and BubR1 phosphorylation in cells treated with Palbo, Abema (5 µM), or alisertib in the presence of MG132, correlating with SAC inactivation. Right: Immunofluorescence shows partial reduction of kinetochore pMELT signals after Palbo or Abema treatment. Scale bar: 10 µm.

Article Snippet: For kinase inhibition, we employed CDK4/6 inhibitors—palbociclib (MCE, HY-50767), abemaciclib (MCE, HY-16297A), and ribociclib (MCE, HY-15777)—as well as the CDK1-specific inhibitor RO3306 (Selleck, 872573-93-8; MCE, HY-12529), the Aurora A kinase inhibitor alisertib (MCE, HY-10971) and the MEK inhibitor PD0325901 (Selleck S1036).).

Techniques: Incubation, Flow Cytometry, Western Blot, Inhibition, Control, Marker, Immunofluorescence, Positive Control, Phospho-proteomics