Journal: bioRxiv

Article Title: PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress

doi: 10.64898/2026.03.16.711577

Figure Lengend Snippet: (A) Experimental setup for steady-state RNA sequencing. (B) Numbers of up- or downregulated genes (log2-fold change >1) after 24 or 48 h HRAS G12V or KRAS G12V induction. (C) Numbers of unique and shared up-regulated genes after HRAS G12V or KRAS G12V induction. (D) Functional enrichment analysis (gene ontology, biological process) of genes downregulated 48 h after HRAS G12V or KRAS G12V induction. (E) Functional enrichment analysis (gene ontology, biological process) of genes upregulated 48 h after HRAS G12V or KRAS G12V induction. (G) Log2 fold-change in hallmark E2F target gene expression after HRAS G12V or KRAS G12V induction. (H) E2F1 expression (RNAseq, DEseq2) after HRAS G12V or KRAS G12V induction. N=3. (I) Log2 fold-change in hallmark MYC target gene expression after HRAS G12V or KRAS G12V induction. (J) MYC expression (RNAseq, DEseq2) after HRAS G12V or KRAS G12V induction. N=3. (K) Protein levels of pRB1 and α-TUBULIN after oncogene induction for the times indicated. (L) S phase percentage after HRAS G12V induction as determined by EdU labelling and flow cytometry. N=4. (M) S phase percentage after KRAS G12V induction. N=4. (N) S phase percentage after BRAF V600E induction. N=4. (O) Experimental setup for CDK4/6 inhibitor (CDK4/6i) treatment and release. (P) S phase percentage after HRAS G12V induction and treatment with CDK4/6i. N=2. (Q) Nuclear EU intensity after HRAS G12V induction and release from CDK4/6i. N=3. (R) Average replication fork speeds after HRAS G12V induction and release from CDK4/6i. N=3. Means +/-SEM (bars) are shown with 2-way ANOVA or mixed effects analysis.

Article Snippet: Small molecule inhibitors and activators were sourced as follows: MEK inhibitor PD0325901 (1 μM) and CDK4/6 inhibitor Palbociclib (1 μM) were obtained from Merck Life Science UK Limited.

Techniques: RNA Sequencing, Functional Assay, Targeted Gene Expression, Expressing, RNA sequencing, Flow Cytometry

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, b, HCT116 cells were synchronised at the G1/S boundary using a double thymidine block and released into nocodazole-containing medium to activate the spindle assembly checkpoint (SAC). Cells were treated with 5 µM palbociclib (Palbo) or vehicle control (+Noc) and harvested at the indicated time points for analysis by flow cytometry. a, Representative flow cytometry profiles showing DNA content (DAPI) and histone H3 serine 10 phosphorylation (pH3) at time points from T0 to T12. b, Quantification of mitotic cells (4N DNA content with positive pH3 signals, left) and G2-like cells (4N DNA content with negative pH3 signals, right). Palbo-treated cells showed reduced mitotic arrest and an increase in the G2-like population. Data are presented as means ± s.d. (N = 3). Statistical significance was assessed using two-way ANOVA. c, Representative immunofluorescence images of multinucleated interphase cells, indicative of mitotic exit following chromosome missegregation, stained for DNA (Hoechst, cyan), β-tubulin (red), and Lamin A/C (green). Yellow dashed circles indicate multinucleated cells. Scale bars, 10 μm. d–f, CDK4/6 inhibition in nocodazole-arrested mitotic cells induces mitotic slippage. d, Schematic of the experimental design and flowcytometry analysis: Cells were synchronised at G1/S, released into nocodazole-containing medium, and incubated for 9 hours to arrest in prometaphase. Palbo was added, and samples were collected at 0, 1, 1.5, 2, 2.5, and 3 hours after addition. e, Flow cytometry analysis of mitotic (4N pH3+) and G2-like (4N pH3−) cell populations over time. Data are presented as means ± s.d. (N = 3). f, ight: Multinucleated cells were quantified and presented as means ± s.d. (N = 3). Statistical significance was assessed using two-way ANOVA. The sample size (n) for each condition and time point across three biological replicates is as follows: replicate 1 (-: 123, 105, 102, 109, 109, 118, 142, 106, 117; +Palbo: 109, 113, 112, 114, 106, 101, 128, 124, 129), replicate 2 (-: 172, 93, 97, 108, 105, 126, 113, 93, 122; +Palbo: 123, 132, 116, 125, 132, 143, 135, 113, 123), replicate 3 (-: 192, 187, 190, 132, 195, 108, 184, 191, 175; +Palbo: 130, 309, 187, 121, 165, 162, 144, 128, 173) at T1 to T5.

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: Blocking Assay, Control, Flow Cytometry, Phospho-proteomics, Immunofluorescence, Staining, Inhibition, Incubation

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

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, Schematic of the experimental workflow for phosphoproteomic analysis. HCT116 cells were synchronised at prometaphase by 9-hour nocodazole and MG132 treatment after thymidine release (T0). Cells were exposed to 1 µM or 5 µM Palbo or control (-) for 2 hours (T2) and harvested for phosphoproteomic analysis. BubR1 dissociation from kinetochores and cell cycle profiles were confirmed as indicators of CDK4/6 inhibition efficiency. b, Summary of identified phosphorylation sites, peptides, and proteins across all samples. Sites and proteins that were comparable between treatments are shown. c, Numbers of differentially phosphorylated sites (upregulated and downregulated) and proteins in 1 µM and 5 µM Palbo-treated cells compared to controls (fold change > 1.5 or < 0.667, p < 0.05). d, Heatmap of global phosphorylation profiles across all samples, showing changes induced by 1 µM and 5 µM Palbo treatment. e, Established CDK4/6 substrates, RB1 and RBL1, showed significant reduction in phosphorylation upon treatment with Palbo treatment. f, Motif enrichment analysis of differentially phosphorylated sites revealed significant enrichment of the pS/T-P motif, characteristic of CDK4/6 substrates. g, Venn diagram illustrating chromosome segregation-related proteins among significantly downregulated phosphoproteins upon 1 µM or/and 5 µM Palbo treatment (fold change < 0.5, p < 0.05). h, Volcano plots showing differentially phosphorylated proteins associated with chromosome segregation in 1 µM and 5 µM Palbo treatments compared to controls. Key SAC-related proteins are highlighted. i, Putative CDK4/6 phosphorylation sites identified on SAC-related proteins, CENP-E, CENP-F, BUB1, ZW10, INCENP, and NUMA1, with significant reduction in phosphorylation upon Palbo treatment.

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: Control, Inhibition, Phospho-proteomics