apc3  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: CCT5 maintains mitotic fidelity and promotes early colorectal tumorigenesis

doi: 10.1016/j.isci.2026.115223

Figure Lengend Snippet: CCT5 maintains the structural integrity of the MCC-APC/C complex and facilitates APC/C activation (A) Co-immunoprecipitation (CoIP) analysis of CDC20-associated proteins follows release from double thymidine block. CDC20 was immunoprecipitated, and the association of mitotic checkpoint complex (MCC) components, including BUBR1, CDC27, BUB3, and MAD2L1, was examined by immunoblotting. Representative immunoblots from n = 3 independent biological experiments are shown (n represents independent experiments). Reduced association of MCC components with CDC20 was observed in CCT5-depleted cells. (B) Co-immunoprecipitation (CoIP) analysis of APC/C-associated proteins follows release from double thymidine block. APC3 was immunoprecipitated, and the association of MCC components, including BUBR1, CDC27, CDC20, BUB3, and MAD2L1, was examined by immunoblotting. Representative immunoblots from n = 3 independent biological experiments are shown (n represents independent experiments). Reduced association of MCC components with the APC/C complex was observed in CCT5-depleted cells, while input protein levels remained comparable. (C and D) Cycloheximide (CHX) chase assay shows reduced stability of CDC20 in CCT5-deficient HCT116 cells. Representative immunoblots are shown in (C). Quantitative densitometric analysis of CDC20 protein levels normalized to GAPDH and expressed relative to the 0-h time point is shown in (D). Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance was determined using two-way ANOVA (group × time) followed by Šídák’s multiple comparisons test. ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. (E) RT-qPCR analysis of CDC20 mRNA expression follows CCT5 knockdown in HCT116 cells. No significant change in CDC20 mRNA levels was observed. Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test. ns, not significant. (F) Cell viability assay assesses whether CDC20 overexpression rescues the proliferation defect induced by CCT5 knockdown in HCT116 cells. Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance was determined using two-way ANOVA (group × time) followed by Šídák’s multiple comparisons test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CDC20 overexpression did not restore proliferation in CCT5-depleted cells. (G and H) Flow cytometry analysis of cell cycle distribution in HCT116 cells follows CCT5 knockdown and CDC20 overexpression. Representative histograms are shown in (G). Quantification of the G2/M fraction is shown in (H). Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance for the G2/M fraction was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. ∗∗ p < 0.01; ns, not significant. CDC20 overexpression did not restore the G2/M phase distribution in CCT5-depleted cells. (I and J) Ubiquitination assay shows increased CDC20 ubiquitination following CCT5 knockdown in HCT116 cells under asynchronous conditions (nocodazole −, MG132 +). CDC20 was immunoprecipitated, and ubiquitinated CDC20 was detected by immunoblotting with anti-ubiquitin antibodies (I). Quantitative densitometric analysis of ubiquitinated CDC20 normalized to immunoprecipitated CDC20 is shown in (J). Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance was assessed using an unpaired t test. ∗∗ p < 0.01. (K and L) Ubiquitination assay shows reduced CDC20 ubiquitination following CCT5 knockdown in HCT116 cells under nocodazole-induced mitotic arrest conditions (NOC +, MG132 +). CDC20 was immunoprecipitated and ubiquitinated. CDC20 was detected by immunoblotting with anti-ubiquitin antibodies (K). Quantitative densitometric analysis of ubiquitinated CDC20 normalized to immunoprecipitated CDC20 is shown in (L). Experiments were performed in n = 3 independent biological replicates (n represents independent experiments). Data are presented as mean ± SD. Statistical significance was assessed using an unpaired t test. ∗ p < 0.05.

Article Snippet: Mouse monoclonal anti-CDC27 (APC3) , Santa Cruz Biotechnology , Cat# sc-9972, RRID: AB_627228.

Techniques: Activation Assay, Immunoprecipitation, Blocking Assay, Western Blot, Quantitative RT-PCR, Expressing, Knockdown, Two Tailed Test, Viability Assay, Over Expression, Flow Cytometry, Ubiquitin Proteomics

Journal: Nature communications

Article Title: APC/C-mediated ubiquitylation of extranucleosomal histone complexes lacking canonical degrons.

doi: 10.1038/s41467-025-57384-7

Figure Lengend Snippet: Fig. 1 | APC3 binds nucleosome acidic patch through HMGN-like nucleosome binding motif. a Electrostatic surface of nucleosome (PDB 3AFA), left, and zoomed view of acidic patch showing residues mutated in acidic patch interaction screen, right. b Profile plots of APC/C subunits and HMGN1 for triplicate wild-type and mutant nucleosome affinity proteomics experiments (data from41). Individual quantitated peptides shown in grey and weighted averages in red or blue. ΔAP: H2A E61A, E64S, N68A, D72S, N89A, D90A, E91S. Additional profile plots shown in Supplementary Fig. 1. c Pulldowns from HEK293T nuclear lysates using recon- stituted, wild-type or mutant biotinylated nucleosomes followed by western blot

Article Snippet: A western blot was later performed with the same membrane using a mouse anti-APC3 antibody (1:10,000, Santa Cruz, SC-9972) and 680CWgoat anti-mouse IgG secondary antibody (1:10,000, LI-COR) allowing simultaneous detection of the label transfer reagent and APC3.

Techniques: Binding Assay, Mutagenesis, Western Blot

Journal: Blood Cancer Discovery

Article Title: Mis-splicing of Mitotic Regulators Sensitizes SF3B1-Mutated Human HSCs to CHK1 Inhibition

doi: 10.1158/2643-3230.BCD-23-0230

Figure Lengend Snippet: SF3B1 -mutant cells have delayed G2/M cell cycle progression. A, EdU cell cycle analysis of WT and SF3B1 K700E K562 cells. Representative flow plots (left) and quantitation (right) of the proportion in S or G2/M phase. Mean ± SD, n = 6 independent experiments, unpaired t test. B, EdU cell cycle analysis of control or SF3B1 K700E edited CB cells expressing HSC marker CD133. Representative flow plots (left) and quantitation (right). Mean ± SD, n = 2 independent experiments, paired t test. C, Proportion of WT and SF3B1 K700E K562 cells positive for pH3, a marker of mitosis. Representative flow plots (left) and quantitation (right). Mean ± SD, of n = 8 experiments, unpaired t test. D, Proportion of pH3-positive mitotic WT and SF3B1 -mutant cells after release from G2/M block using CDK1 inhibitor RO-3306. n = 2 independent time-course experiments, unpaired t test. E, Mis-splicing of BUBR1 and CDC27 in SF3B1 WT or mutant (MUT) MDS patients, iPSC-HSPCs, K562 cells, and edited CB/PB CD34 + HSPCs, or normal bone marrow (BM); significance shown using 1-sided Mann–Whitney U test. F, Western Blot analysis of BUBR1 (top) and CDC27 (bottom) protein level in SF3B1 -mutant (MUT) and WT K562 cells. Expression normalized to GAPDH and shown as fold change relative to WT; n = 3 experiments, mean ± SD. G, EdU cell cycle analysis of WT K562 cells transduced with control luciferase (WT), BUBR1 (B2, B3), or CDC27 (C2, C3) shRNAs, or SF3B1 -mutant K562 cells transduced with control shRNA (MUT). Left: representative EdU cell cycle flow plots. Right: proportion of BUBR1 (left) or CDC27 (right) knockdown K562 cells in G2/M phase. Mean ± SD, of n = 4 experiments; unpaired t test.

Article Snippet: Lysates were resolved by 4% to 20% SDS-PAGE (Bio-Rad) and immunoblotted with antibodies for GAPDH (1:10,000, Abcam, ab9485), HSP90 (1:10,000, BD Biosciences #610419), RUNX1 (1:1,000, Santa Cruz sc-365644), STAG2 (1:1,000, Santa Cruz sc-81852), BUBR1 (1:1,000, BD Biosciences #612502), CDC27 (1:500, Cell Signaling Technologies #12530), pCHK1 S345 (1:1,000, Cell Signaling Technologies #2348), CHK1 (1:1,000, Cell Signalling Technology #2360), DYNLL1 (1:5,000, abcam #51603).

Techniques: Mutagenesis, Cell Cycle Assay, Quantitation Assay, Control, Expressing, Marker, Blocking Assay, MANN-WHITNEY, Western Blot, Transduction, Luciferase, shRNA, Knockdown

Journal: Blood Cancer Discovery

Article Title: Mis-splicing of Mitotic Regulators Sensitizes SF3B1-Mutated Human HSCs to CHK1 Inhibition

doi: 10.1158/2643-3230.BCD-23-0230

Figure Lengend Snippet: SF3B1 -mutant cells are selectively sensitized to CHK1 inhibition. A–C, IC 50 value of CHK1 inhibitor AZD-7762 ( A ), CHK1 inhibitor prexasertib LY2606368 ( B ), or SF3b inhibitor pladienolide B ( C ) in WT (Ctrl) or SF3B1 -mutant (S-A, S-R, S-S) K562 cells. Cells were treated with drugs in a dose-response format for 5 days. Mean ± SD, of four independent experiments, one-way ANOVA with Dunnett’s correction for multiple comparisons. D, CHK1 phosphorylation in WT (Ctrl) or SF3B1 -mutant (S-A, S-R, S-S) K562 cells. Left: Western blot analysis of pCHK1 (S345) and total CHK1 treated for 5 hours with DMSO or 10 nmol/L CHK1i prexasertib. Right: Ratio of pCHK1 to total CHK1 in SF3B1 -mutant normalized to WT cells. n = 2 independent experiments, two technical replicates. E, Cell growth of WT and SF3B1 -mutant K562 cells after CRISPR-mediated ablation of CHK1 . Left: CHK1 protein level after control AAVS1 (sgCtrl) or CHK1 (sgCHK1) knockout. Right: Cell growth of WT and SF3B1 -mutant cells with sgCHK1 or sgCtrl at 3–6 days of culture, relative to day 0 (72 hours after editing). n = 3 independent experiments, ratio paired t test. F, Number of CB-derived control (Ctrl) or SF3B1 K700E-edited (S-A, S-R, S-S) CD34 + HSPCs after treatment with 2.5 nmol/L pladienolide B (right) or 2.5 nmol/L CHK1i prexasertib (left) for 7 days. Fold change of CD34 + cell number after drug treatment was calculated relative to vehicle (DMSO). Mean ± SD, of n = 3 independent experiments; paired t test. G, IC 50 value of CHK1i prexasertib in WT K562 cells transduced with control luciferase (WT), BUBR1 (B2, B3), or CDC27 (C2, C3) shRNAs, or SF3B1 -mutant K562 cells transduced with control shRNA (MUT). Cells were treated with prexasertib in a dose response format for 5 days; two independent shRNAs, two sets of lines independently generated for each condition. Mean ± SD, of three independent experiments, one-way ANOVA with Dunnett’s correction for multiple comparisons.

Article Snippet: Lysates were resolved by 4% to 20% SDS-PAGE (Bio-Rad) and immunoblotted with antibodies for GAPDH (1:10,000, Abcam, ab9485), HSP90 (1:10,000, BD Biosciences #610419), RUNX1 (1:1,000, Santa Cruz sc-365644), STAG2 (1:1,000, Santa Cruz sc-81852), BUBR1 (1:1,000, BD Biosciences #612502), CDC27 (1:500, Cell Signaling Technologies #12530), pCHK1 S345 (1:1,000, Cell Signaling Technologies #2348), CHK1 (1:1,000, Cell Signalling Technology #2360), DYNLL1 (1:5,000, abcam #51603).

Techniques: Mutagenesis, Inhibition, Phospho-proteomics, Western Blot, CRISPR, Control, Knock-Out, Derivative Assay, Transduction, Luciferase, shRNA, Generated

Journal: Blood Cancer Discovery

Article Title: Mis-splicing of Mitotic Regulators Sensitizes SF3B1-Mutated Human HSCs to CHK1 Inhibition

doi: 10.1158/2643-3230.BCD-23-0230

Figure Lengend Snippet: CHK1 inhibition selectively targets SF3B1 -mutant HSCs. A, Experimental strategy to determine the number of WT (Ctrl) or SF3B1 K700E-edited (BFP + ) CD34 + CD133 + (CD38 − ) phenotypic HSCs after drug treatment; BV605: empty channel. B, Representative flow plots of the % CD34 + CD133 + cells after 7 days of treatment with vehicle (DMSO), 2.5 nmol/L pladienolide B, or 2.5 nmol/L prexasertib. C, Number of CD34 + CD133 + phenotypic HSCs edited for control (Ctrl) or SF3B1 K700E (S-A, S-R, S-S) after 7 days of treatment with 2.5 nmol/L prexasertib. CD34 + cells derived from CB (left) or PB (right) donors. Fold change of CD34 + CD133 + phenotypic HSC number after drug treatment was calculated for each genotype, relative to vehicle (DMSO). Mean ± SD, of n = 3 independent experiments; *, P < 0.05; **, P < 0.01; paired t test. D, Same as C with 2.5 nmol/L pladienolide B (CB). E, Number of normal CB CD34 + CD133 + phenotypic HSCs transduced with control or CDC27 shRNA after 7 days of treatment with 2.5 nmol/L prexasertib. Mean ± SD, of n = 2 independent experiments; paired t test. F, Experimental approach for in vivo treatment with prexasertib CHK1i. NSG-SGM3 mice were transplanted with SF3B1 / RUNX1 mutant MDS patient iPSC-derived HSPCs and treated with vehicle (Captisol) or 10 mg/kg prexasertib hydrochloride for 3 weeks. G, Engraftment of SF3B1 / RUNX1 mutant iPSC-HSPCs in NSG-SGM3 mice treated with vehicle or CHK1i for 3 weeks. Right: representative flow plots and gating strategy to determine human engraftment. Human engraftment was determined as % of human CD45 + CD33 + cells in the injected femur 7 weeks after transplantation. Mean ± SD, of n = 6 vehicle and n = 6 CHK1i mice, unpaired t test. H, Experimental approach for in vivo treatment with prexasertib CHK1i of mice co-transplanted with SF3B1 -WT and mutant cells. NSG-SGM3 mice were transplanted with SF3B1 -WT/ RUNX1 -mutant and SF3B1 / RUNX1 double-mutant iPSC-HSPCs in a 1:1 ratio and treated with vehicle (Captisol) or 10 mg/kg prexasertib hydrochloride for 3 weeks. I, Percentage of SF3B1 -mutant iPSC-HSPCs of human engraftment in NSG-SGM3 mice co-transplanted with SF3B1 -WT and mutant cells and treated with vehicle or CHK1i for 3 weeks. Human CD45 + CD33 + cells were isolated from individual mice and % of SF3B1 K700E cells determined by amplicon next generation sequencing. Mean ± SD, of n = 3 vehicle and n = 3 CHK1i mice, unpaired t test.

Article Snippet: Lysates were resolved by 4% to 20% SDS-PAGE (Bio-Rad) and immunoblotted with antibodies for GAPDH (1:10,000, Abcam, ab9485), HSP90 (1:10,000, BD Biosciences #610419), RUNX1 (1:1,000, Santa Cruz sc-365644), STAG2 (1:1,000, Santa Cruz sc-81852), BUBR1 (1:1,000, BD Biosciences #612502), CDC27 (1:500, Cell Signaling Technologies #12530), pCHK1 S345 (1:1,000, Cell Signaling Technologies #2348), CHK1 (1:1,000, Cell Signalling Technology #2360), DYNLL1 (1:5,000, abcam #51603).

Techniques: Inhibition, Mutagenesis, Control, Derivative Assay, Transduction, shRNA, In Vivo, Injection, Transplantation Assay, Isolation, Amplification, Next-Generation Sequencing

Journal: Blood Cancer Discovery

Article Title: Mis-splicing of Mitotic Regulators Sensitizes SF3B1-Mutated Human HSCs to CHK1 Inhibition

doi: 10.1158/2643-3230.BCD-23-0230

Figure Lengend Snippet:

Article Snippet: Lysates were resolved by 4% to 20% SDS-PAGE (Bio-Rad) and immunoblotted with antibodies for GAPDH (1:10,000, Abcam, ab9485), HSP90 (1:10,000, BD Biosciences #610419), RUNX1 (1:1,000, Santa Cruz sc-365644), STAG2 (1:1,000, Santa Cruz sc-81852), BUBR1 (1:1,000, BD Biosciences #612502), CDC27 (1:500, Cell Signaling Technologies #12530), pCHK1 S345 (1:1,000, Cell Signaling Technologies #2348), CHK1 (1:1,000, Cell Signalling Technology #2360), DYNLL1 (1:5,000, abcam #51603).

Techniques: Control, Luciferase