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CWC22 knockdown causes mitotic slippage <t>through</t> <t>APC/C</t> . MIA PaCa-2 cells were transfected with control siRNA (siControl) or CWC22-targeting siRNAs (siCWC22#1 and #2). A and B , at 39 h after transfection, the cells were treated with 5 μM STLC for 1 h and monitored for 24 h by time-lapse imaging with 0.1 μM Hoechst 33342. A , the duration of each mitotic phase is shown as indicated in D . In total, 20 mitotic cells were examined. B , the duration of mitosis until mitotic slippage ( red ) or cell death ( yellow ) was measured among the cells that entered mitosis within 4 h of starting time-lapse analysis is plotted as the mean ± SD from two experiments (n ≥ 37). Prometaphase-arrested cells where no events occurred over 20 h ( green ) are included in ≥20 h. C , at 40 h after siRNA transfection, the cells were fixed and stained for cyclin B1 ( green ) and DNA ( red ). Representative images are shown, and yellow arrowheads show the representative cells. The scale bars represent 10 μm. The mean fluorescence intensity of cyclin B1 within whole cells in prophase or prometaphase per cell was measured and plotted as the mean ± SD from a representative experiment (n = 20). D , at 36 h after transfection, cells were treated with 5 μM STLC for 12 h. The mitotic cells were collected via mitotic shake-off, and Western blot analysis was performed with the indicated antibodies. An asterisk shows a nonspecific band. E , at 36 h after transfection, cells were treated with 5 μM STLC with or without 5 μM <t>proTAME</t> for 12 h. The mitotic cells were collected via mitotic shake-off, and Western blot analysis was performed with the indicated antibodies. An asterisk shows a nonspecific band. F and G , at 39 h after transfection, the cells were treated with 5 μM STLC in the presence or absence of 5 μM proTAME for 1 h and monitored for 24 h by time-lapse imaging with 0.1 μM Hoechst 33342. F , the duration of each mitotic phase is shown as indicated in A . In total, 20 mitotic cells were examined. G , the duration of mitosis is shown as indicated in B (n ≥ 33). P Values were determined using the Steel test in B , the Games–Howell test in C , and the Steel–Dwass test in G . APC/C, anaphase-promoting complex/cyclosome; STLC, S-trityl- l -cysteine.

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1) Product Images from "Targeting the splicing factor CWC22 induces mitotic slippage through repression of BubR1 expression and CDK1 activity in cancer cells"

Article Title: Targeting the splicing factor CWC22 induces mitotic slippage through repression of BubR1 expression and CDK1 activity in cancer cells

Journal: The Journal of Biological Chemistry

doi: 10.1016/j.jbc.2026.111148

CWC22 knockdown causes mitotic slippage through APC/C . MIA PaCa-2 cells were transfected with control siRNA (siControl) or CWC22-targeting siRNAs (siCWC22#1 and #2). A and B , at 39 h after transfection, the cells were treated with 5 μM STLC for 1 h and monitored for 24 h by time-lapse imaging with 0.1 μM Hoechst 33342. A , the duration of each mitotic phase is shown as indicated in D . In total, 20 mitotic cells were examined. B , the duration of mitosis until mitotic slippage ( red ) or cell death ( yellow ) was measured among the cells that entered mitosis within 4 h of starting time-lapse analysis is plotted as the mean ± SD from two experiments (n ≥ 37). Prometaphase-arrested cells where no events occurred over 20 h ( green ) are included in ≥20 h. C , at 40 h after siRNA transfection, the cells were fixed and stained for cyclin B1 ( green ) and DNA ( red ). Representative images are shown, and yellow arrowheads show the representative cells. The scale bars represent 10 μm. The mean fluorescence intensity of cyclin B1 within whole cells in prophase or prometaphase per cell was measured and plotted as the mean ± SD from a representative experiment (n = 20). D , at 36 h after transfection, cells were treated with 5 μM STLC for 12 h. The mitotic cells were collected via mitotic shake-off, and Western blot analysis was performed with the indicated antibodies. An asterisk shows a nonspecific band. E , at 36 h after transfection, cells were treated with 5 μM STLC with or without 5 μM proTAME for 12 h. The mitotic cells were collected via mitotic shake-off, and Western blot analysis was performed with the indicated antibodies. An asterisk shows a nonspecific band. F and G , at 39 h after transfection, the cells were treated with 5 μM STLC in the presence or absence of 5 μM proTAME for 1 h and monitored for 24 h by time-lapse imaging with 0.1 μM Hoechst 33342. F , the duration of each mitotic phase is shown as indicated in A . In total, 20 mitotic cells were examined. G , the duration of mitosis is shown as indicated in B (n ≥ 33). P Values were determined using the Steel test in B , the Games–Howell test in C , and the Steel–Dwass test in G . APC/C, anaphase-promoting complex/cyclosome; STLC, S-trityl- l -cysteine.

Figure Legend Snippet: CWC22 knockdown causes mitotic slippage through APC/C . MIA PaCa-2 cells were transfected with control siRNA (siControl) or CWC22-targeting siRNAs (siCWC22#1 and #2). A and B , at 39 h after transfection, the cells were treated with 5 μM STLC for 1 h and monitored for 24 h by time-lapse imaging with 0.1 μM Hoechst 33342. A , the duration of each mitotic phase is shown as indicated in D . In total, 20 mitotic cells were examined. B , the duration of mitosis until mitotic slippage ( red ) or cell death ( yellow ) was measured among the cells that entered mitosis within 4 h of starting time-lapse analysis is plotted as the mean ± SD from two experiments (n ≥ 37). Prometaphase-arrested cells where no events occurred over 20 h ( green ) are included in ≥20 h. C , at 40 h after siRNA transfection, the cells were fixed and stained for cyclin B1 ( green ) and DNA ( red ). Representative images are shown, and yellow arrowheads show the representative cells. The scale bars represent 10 μm. The mean fluorescence intensity of cyclin B1 within whole cells in prophase or prometaphase per cell was measured and plotted as the mean ± SD from a representative experiment (n = 20). D , at 36 h after transfection, cells were treated with 5 μM STLC for 12 h. The mitotic cells were collected via mitotic shake-off, and Western blot analysis was performed with the indicated antibodies. An asterisk shows a nonspecific band. E , at 36 h after transfection, cells were treated with 5 μM STLC with or without 5 μM proTAME for 12 h. The mitotic cells were collected via mitotic shake-off, and Western blot analysis was performed with the indicated antibodies. An asterisk shows a nonspecific band. F and G , at 39 h after transfection, the cells were treated with 5 μM STLC in the presence or absence of 5 μM proTAME for 1 h and monitored for 24 h by time-lapse imaging with 0.1 μM Hoechst 33342. F , the duration of each mitotic phase is shown as indicated in A . In total, 20 mitotic cells were examined. G , the duration of mitosis is shown as indicated in B (n ≥ 33). P Values were determined using the Steel test in B , the Games–Howell test in C , and the Steel–Dwass test in G . APC/C, anaphase-promoting complex/cyclosome; STLC, S-trityl- l -cysteine.

Techniques Used: Knockdown, Transfection, Control, Imaging, Staining, Fluorescence, Western Blot

CWC22 knockdown downregulates the mRNA levels of SAC-regulatory genes, including BubR1 and Bub1 . A – E , MIA PaCa-2/shCWC22#2 cells were treated with 4 μg/ml Dox for 40 h and then treated with 5 μM STLC plus 5 μM proTAME for 16 h. Mitotic cells were collected by mitotic shake-off and analyzed by RNA-Seq. A , a schematic depiction of the synchronization method is shown. B and C , volcano plot showing differential degree of intron retention ( B ) or differential gene expression ( C ) in Dox-treated cells compared with Dox-untreated cells. Representative upregulated and downregulated genes are labeled in each panel. D , GSEA plots for the gene set “REACTOME_MITOTIC_SPINDLE_CHECKPOINT” between Dox-treated and untreated cells are shown. E , heatmap of relative expression values (z-score of normalized read counts) from REACTOME_MITOTIC_SPINDLE_CHECKPOINT dataset (n = 2). F , MIA PaCa-2 cells were transfected with control siRNA (siControl) or CWC22-targeting siRNAs (siCWC22#1 and #2). At 44 h after transfection, the cells were treated with 5 μM STLC plus 5 μM proTAME for 16 h. The mitotic cells were collected via mitotic shake-off and analyzed by real-time PCR for BUB1B and BUB1 mRNA. The fold changes in mRNA levels are expressed relative to siControl. P Values were determined using the Audic and Claverie test in B , the Wald's test in C , and the Games–Howell test in F . Dox, doxycycline; FDR, false discovery rate q value; GSEA, gene set enrichment analysis; NES, normalized enrichment score; SAC, spindle assembly checkpoint; STLC, S-trityl- l -cysteine.

Figure Legend Snippet: CWC22 knockdown downregulates the mRNA levels of SAC-regulatory genes, including BubR1 and Bub1 . A – E , MIA PaCa-2/shCWC22#2 cells were treated with 4 μg/ml Dox for 40 h and then treated with 5 μM STLC plus 5 μM proTAME for 16 h. Mitotic cells were collected by mitotic shake-off and analyzed by RNA-Seq. A , a schematic depiction of the synchronization method is shown. B and C , volcano plot showing differential degree of intron retention ( B ) or differential gene expression ( C ) in Dox-treated cells compared with Dox-untreated cells. Representative upregulated and downregulated genes are labeled in each panel. D , GSEA plots for the gene set “REACTOME_MITOTIC_SPINDLE_CHECKPOINT” between Dox-treated and untreated cells are shown. E , heatmap of relative expression values (z-score of normalized read counts) from REACTOME_MITOTIC_SPINDLE_CHECKPOINT dataset (n = 2). F , MIA PaCa-2 cells were transfected with control siRNA (siControl) or CWC22-targeting siRNAs (siCWC22#1 and #2). At 44 h after transfection, the cells were treated with 5 μM STLC plus 5 μM proTAME for 16 h. The mitotic cells were collected via mitotic shake-off and analyzed by real-time PCR for BUB1B and BUB1 mRNA. The fold changes in mRNA levels are expressed relative to siControl. P Values were determined using the Audic and Claverie test in B , the Wald's test in C , and the Games–Howell test in F . Dox, doxycycline; FDR, false discovery rate q value; GSEA, gene set enrichment analysis; NES, normalized enrichment score; SAC, spindle assembly checkpoint; STLC, S-trityl- l -cysteine.

Techniques Used: Knockdown, RNA Sequencing, Gene Expression, Labeling, Expressing, Transfection, Control, Real-time Polymerase Chain Reaction

Targeting the highly expressed CWC22 strongly decreases the viable number in cancers . A , the relative mRNA expression of CWC22 in normal pancreatic tissue and pancreatic ductal adenocarcinoma (PDAC) tissue ( left ) and normal cervical tissue and cervical squamous cell carcinoma (CESC) tissue ( right ) in GSE60979 and GSE7410 datasets are shown, respectively. B , pancreatic adenocarcinoma (PAAD) patients ( left ) and CESC patients ( right ) in TCGA datasets were divided into two groups based on CWC22 expression level, and Kaplan–Meier curves for overall survival are shown. C , MIA PaCa-2 cells or HeLa S3 cells were transfected with control siRNA (siControl) or CWC22-targeting siRNAs (siCWC22#1 and #2). After siRNA transfection, the cells were further cultured for 5 days. Cell numbers were counted 2, 4, and 6 days after seeding, and viable cell numbers were plotted. D , model of the role of CWC22 in cell cycle progression in cancer cells. Control cells exhibit proper cell cycle progression. CWC22 knockdown prolongs G2 phase duration because of the DNA damage accumulation. CWC22-knockdown cells entering mitosis undergo defects in chromosome alignment and subsequent mitotic slippage because of the APC/C activation and CDK1 inactivation through both BubR1 downregulation and accumulation of inhibitory phosphorylation of CDK1. Eventually, cells undergoing prolonged interphase or mitotic slippage exhibit cell death. APC/C, anaphase-promoting complex/cyclosome; CDK1, cyclin-dependent kinase 1; TCGA, The Cancer Genome Atlas.

Figure Legend Snippet: Targeting the highly expressed CWC22 strongly decreases the viable number in cancers . A , the relative mRNA expression of CWC22 in normal pancreatic tissue and pancreatic ductal adenocarcinoma (PDAC) tissue ( left ) and normal cervical tissue and cervical squamous cell carcinoma (CESC) tissue ( right ) in GSE60979 and GSE7410 datasets are shown, respectively. B , pancreatic adenocarcinoma (PAAD) patients ( left ) and CESC patients ( right ) in TCGA datasets were divided into two groups based on CWC22 expression level, and Kaplan–Meier curves for overall survival are shown. C , MIA PaCa-2 cells or HeLa S3 cells were transfected with control siRNA (siControl) or CWC22-targeting siRNAs (siCWC22#1 and #2). After siRNA transfection, the cells were further cultured for 5 days. Cell numbers were counted 2, 4, and 6 days after seeding, and viable cell numbers were plotted. D , model of the role of CWC22 in cell cycle progression in cancer cells. Control cells exhibit proper cell cycle progression. CWC22 knockdown prolongs G2 phase duration because of the DNA damage accumulation. CWC22-knockdown cells entering mitosis undergo defects in chromosome alignment and subsequent mitotic slippage because of the APC/C activation and CDK1 inactivation through both BubR1 downregulation and accumulation of inhibitory phosphorylation of CDK1. Eventually, cells undergoing prolonged interphase or mitotic slippage exhibit cell death. APC/C, anaphase-promoting complex/cyclosome; CDK1, cyclin-dependent kinase 1; TCGA, The Cancer Genome Atlas.

Techniques Used: Expressing, Transfection, Control, Cell Culture, Knockdown, Activation Assay, Phospho-proteomics

other:

Article Title: Targeting the splicing factor CWC22 induces mitotic slippage through repression of BubR1 expression and CDK1 activity in cancer cells
Article Snippet: The APC/C inhibitor proTAME (HY-124955; MedChemExpress; SML3977; MilliporeSigma) was used at 5 μM.

Cell Culture:

Article Title: A parent-of-origin effect on embryonic telomere elongation determines telomere length inheritance
Article Snippet: Embryos were cultured in EmbryoMax Advanced KSOM (AKSOM, Millipore Sigma, MR-101-D) in a humidified atmosphere of 5% CO 2 in air at 37 °C. .. Embryos were cultured for 17 hours in AKSOM supplemented with the kinesin-5 inhibitor STLC (S-trityl-L-cysteine, Sigma, 10 μM) and the APC/C inhibitor proTAME (Medchemexpress, 5 μM) either 4 or 24 hours after collection for arrest at the one- or two-cell mitosis stage, respectively. ..

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Journal: The Journal of Biological Chemistry

Article Title: Targeting the splicing factor CWC22 induces mitotic slippage through repression of BubR1 expression and CDK1 activity in cancer cells

doi: 10.1016/j.jbc.2026.111148

Figure Lengend Snippet: CWC22 knockdown causes mitotic slippage through APC/C . MIA PaCa-2 cells were transfected with control siRNA (siControl) or CWC22-targeting siRNAs (siCWC22#1 and #2). A and B , at 39 h after transfection, the cells were treated with 5 μM STLC for 1 h and monitored for 24 h by time-lapse imaging with 0.1 μM Hoechst 33342. A , the duration of each mitotic phase is shown as indicated in D . In total, 20 mitotic cells were examined. B , the duration of mitosis until mitotic slippage ( red ) or cell death ( yellow ) was measured among the cells that entered mitosis within 4 h of starting time-lapse analysis is plotted as the mean ± SD from two experiments (n ≥ 37). Prometaphase-arrested cells where no events occurred over 20 h ( green ) are included in ≥20 h. C , at 40 h after siRNA transfection, the cells were fixed and stained for cyclin B1 ( green ) and DNA ( red ). Representative images are shown, and yellow arrowheads show the representative cells. The scale bars represent 10 μm. The mean fluorescence intensity of cyclin B1 within whole cells in prophase or prometaphase per cell was measured and plotted as the mean ± SD from a representative experiment (n = 20). D , at 36 h after transfection, cells were treated with 5 μM STLC for 12 h. The mitotic cells were collected via mitotic shake-off, and Western blot analysis was performed with the indicated antibodies. An asterisk shows a nonspecific band. E , at 36 h after transfection, cells were treated with 5 μM STLC with or without 5 μM proTAME for 12 h. The mitotic cells were collected via mitotic shake-off, and Western blot analysis was performed with the indicated antibodies. An asterisk shows a nonspecific band. F and G , at 39 h after transfection, the cells were treated with 5 μM STLC in the presence or absence of 5 μM proTAME for 1 h and monitored for 24 h by time-lapse imaging with 0.1 μM Hoechst 33342. F , the duration of each mitotic phase is shown as indicated in A . In total, 20 mitotic cells were examined. G , the duration of mitosis is shown as indicated in B (n ≥ 33). P Values were determined using the Steel test in B , the Games–Howell test in C , and the Steel–Dwass test in G . APC/C, anaphase-promoting complex/cyclosome; STLC, S-trityl- l -cysteine.

Article Snippet: The APC/C inhibitor proTAME (HY-124955; MedChemExpress; SML3977; MilliporeSigma) was used at 5 μM.

Techniques: Knockdown, Transfection, Control, Imaging, Staining, Fluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Targeting the splicing factor CWC22 induces mitotic slippage through repression of BubR1 expression and CDK1 activity in cancer cells

doi: 10.1016/j.jbc.2026.111148

Figure Lengend Snippet: CWC22 knockdown downregulates the mRNA levels of SAC-regulatory genes, including BubR1 and Bub1 . A – E , MIA PaCa-2/shCWC22#2 cells were treated with 4 μg/ml Dox for 40 h and then treated with 5 μM STLC plus 5 μM proTAME for 16 h. Mitotic cells were collected by mitotic shake-off and analyzed by RNA-Seq. A , a schematic depiction of the synchronization method is shown. B and C , volcano plot showing differential degree of intron retention ( B ) or differential gene expression ( C ) in Dox-treated cells compared with Dox-untreated cells. Representative upregulated and downregulated genes are labeled in each panel. D , GSEA plots for the gene set “REACTOME_MITOTIC_SPINDLE_CHECKPOINT” between Dox-treated and untreated cells are shown. E , heatmap of relative expression values (z-score of normalized read counts) from REACTOME_MITOTIC_SPINDLE_CHECKPOINT dataset (n = 2). F , MIA PaCa-2 cells were transfected with control siRNA (siControl) or CWC22-targeting siRNAs (siCWC22#1 and #2). At 44 h after transfection, the cells were treated with 5 μM STLC plus 5 μM proTAME for 16 h. The mitotic cells were collected via mitotic shake-off and analyzed by real-time PCR for BUB1B and BUB1 mRNA. The fold changes in mRNA levels are expressed relative to siControl. P Values were determined using the Audic and Claverie test in B , the Wald's test in C , and the Games–Howell test in F . Dox, doxycycline; FDR, false discovery rate q value; GSEA, gene set enrichment analysis; NES, normalized enrichment score; SAC, spindle assembly checkpoint; STLC, S-trityl- l -cysteine.

Article Snippet: The APC/C inhibitor proTAME (HY-124955; MedChemExpress; SML3977; MilliporeSigma) was used at 5 μM.

Techniques: Knockdown, RNA Sequencing, Gene Expression, Labeling, Expressing, Transfection, Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Targeting the splicing factor CWC22 induces mitotic slippage through repression of BubR1 expression and CDK1 activity in cancer cells

doi: 10.1016/j.jbc.2026.111148

Figure Lengend Snippet: Targeting the highly expressed CWC22 strongly decreases the viable number in cancers . A , the relative mRNA expression of CWC22 in normal pancreatic tissue and pancreatic ductal adenocarcinoma (PDAC) tissue ( left ) and normal cervical tissue and cervical squamous cell carcinoma (CESC) tissue ( right ) in GSE60979 and GSE7410 datasets are shown, respectively. B , pancreatic adenocarcinoma (PAAD) patients ( left ) and CESC patients ( right ) in TCGA datasets were divided into two groups based on CWC22 expression level, and Kaplan–Meier curves for overall survival are shown. C , MIA PaCa-2 cells or HeLa S3 cells were transfected with control siRNA (siControl) or CWC22-targeting siRNAs (siCWC22#1 and #2). After siRNA transfection, the cells were further cultured for 5 days. Cell numbers were counted 2, 4, and 6 days after seeding, and viable cell numbers were plotted. D , model of the role of CWC22 in cell cycle progression in cancer cells. Control cells exhibit proper cell cycle progression. CWC22 knockdown prolongs G2 phase duration because of the DNA damage accumulation. CWC22-knockdown cells entering mitosis undergo defects in chromosome alignment and subsequent mitotic slippage because of the APC/C activation and CDK1 inactivation through both BubR1 downregulation and accumulation of inhibitory phosphorylation of CDK1. Eventually, cells undergoing prolonged interphase or mitotic slippage exhibit cell death. APC/C, anaphase-promoting complex/cyclosome; CDK1, cyclin-dependent kinase 1; TCGA, The Cancer Genome Atlas.

Article Snippet: The APC/C inhibitor proTAME (HY-124955; MedChemExpress; SML3977; MilliporeSigma) was used at 5 μM.

Techniques: Expressing, Transfection, Control, Cell Culture, Knockdown, Activation Assay, Phospho-proteomics

Journal: Nature Communications

Article Title: A prometaphase mechanism of securin destruction is essential for meiotic progression in mouse oocytes

doi: 10.1038/s41467-021-24554-2

Figure Lengend Snippet: a Mean VFP-tagged securin FL (purple dashed trace, n = 11) destruction profile following incubation in 1.5 µM ProTAME to inhibit APC/C activity. Mean VFP-tagged securin FL (magenta trace, n = 16) destruction profile in control oocytes is included as a reference. ProTAME treated oocytes do not extrude a polar body and are therefore aligned at GVBD. b Schematic showing VFP-tagged securin and cyclin B1 constructs. c Mean securin FL (magenta trace, n = 25), securin N101 (pink dashed trace, n = 23), cyclin B1 FL (blue trace, n = 33) and cyclin B1 N90 (light blue dashed trace, n = 34) destruction profiles relative to PB1 extrusion. d Full sequence alignment conservation annotation and multiple sequence alignment of residues 109–133 in securin orthologs. e Mean VFP-tagged securin FL (magenta trace, n = 25), securin N101 (pink dashed trace, n = 23) and securin Δ109–133 (light blue trace, n = 23) destruction profiles relative to PB1 extrusion. f Schematic showing the position of Securin FF-A amino-acid substitutions. Residues F125 and F128 (shown in green in the wild-type protein) were switched to alanines (shown in red in Securin FF-A). g Mean VFP-tagged securin FL (magenta trace, n = 25), securin N101 (pink dashed trace, n = 23) and securin FF-A (green trace, n = 20) destruction traces relative to PB1 extrusion. h Mean VFP-tagged securin FL (magenta dashed trace, n = 23) and securin FF-A (dark green dashed trace, n = 30) destruction profiles following incubation in 150 nM nocodazole to arrest oocytes in prometaphase. Mean VFP-tagged securin FL (magenta trace, n = 16) and securin FF-A (green trace, n = 16) destruction profiles in control oocytes are included as a reference. Nocodazole treated oocytes do not extrude a polar body and are therefore aligned at GVBD. i Mean VFP-tagged securin FL (purple dashed trace, n = 16) and securin FF-A (dark green dashed trace, n = 16) destruction profiles following incubation in 100 nM reversine to block assembly of new SAC complexes. Mean VFP-tagged securin FL (magenta trace, n = 16) and securin FF-A (green trace, n = 16) destruction profiles in control oocytes are included as a reference. Reversine treated oocytes have an accelerated progression through meiosis and are therefore aligned at GVBD. All n numbers refer to the number of individual oocytes analysed over a minimum of three independent experiments. All error bars ± SEM.

Article Snippet: Where necessary, and at the times indicated, nocodazole (Sigma) was added to the media at a final concentration of 150 nM, Mps1 inhibitor reversine (Sigma) at 100 nM , the APC/C inhibitor ProTAME (Boston Biochem) at 1.5 μM, and cycloheximide (Sigma) at 10 μg/ml .

Techniques: Incubation, Activity Assay, Control, Construct, Sequencing, Blocking Assay

Quantification of the percentage of multipolar anaphases following a combination of siRNA treatment and small‐molecule inhibition of dynein (ciliobrevin D: Cilio) or HSET (CW069: CW). Mean ± SEM from a minimum of three independent experiments. **** P < 0.0001; * P < 0.05; ns: non‐significant; unpaired t ‐test. For each condition, a minimum of 600 cells were quantified. Schematic of experimental timeline for DLD‐1 cells used in panels (C–F). Cells were arrested at metaphase‐to‐anaphase transition using proTAME and APCIN APC/C inhibitors. Cells were also treated with or without auxin in combination with small‐molecule inhibitors. Representative immunofluorescence image of DLD‐1 cells with supernumerary centrosomes in control condition. Scale bar in magnified box, 2 μm. Representative immunofluorescence image of DLD‐1 cells with supernumerary centrosomes and depleted of endogenous IFT88‐AID‐YFP upon auxin addition. Scale bar in magnified box, 2 μm. Immunoblots showing endogenous IFT88‐AID‐YFP depletion upon auxin treatment combined or not with small‐molecule inhibitors. Quantification of the percentage of unclustered centrosomes following combination of auxin treatment and small‐molecule inhibition of dynein (Cilio, left panel) or HSET (CW, right panel). Mean ± SEM from a minimum of three independent experiments. ** P < 0.01; ns: non‐significant; unpaired t ‐test. For each condition, a minimum of 233 cells were quantified. Source data are available online for this figure.

Journal: EMBO Reports

Article Title: IFT proteins interact with HSET to promote supernumerary centrosome clustering in mitosis

doi: 10.15252/embr.201949234

Figure Lengend Snippet: Quantification of the percentage of multipolar anaphases following a combination of siRNA treatment and small‐molecule inhibition of dynein (ciliobrevin D: Cilio) or HSET (CW069: CW). Mean ± SEM from a minimum of three independent experiments. **** P < 0.0001; * P < 0.05; ns: non‐significant; unpaired t ‐test. For each condition, a minimum of 600 cells were quantified. Schematic of experimental timeline for DLD‐1 cells used in panels (C–F). Cells were arrested at metaphase‐to‐anaphase transition using proTAME and APCIN APC/C inhibitors. Cells were also treated with or without auxin in combination with small‐molecule inhibitors. Representative immunofluorescence image of DLD‐1 cells with supernumerary centrosomes in control condition. Scale bar in magnified box, 2 μm. Representative immunofluorescence image of DLD‐1 cells with supernumerary centrosomes and depleted of endogenous IFT88‐AID‐YFP upon auxin addition. Scale bar in magnified box, 2 μm. Immunoblots showing endogenous IFT88‐AID‐YFP depletion upon auxin treatment combined or not with small‐molecule inhibitors. Quantification of the percentage of unclustered centrosomes following combination of auxin treatment and small‐molecule inhibition of dynein (Cilio, left panel) or HSET (CW, right panel). Mean ± SEM from a minimum of three independent experiments. ** P < 0.01; ns: non‐significant; unpaired t ‐test. For each condition, a minimum of 233 cells were quantified. Source data are available online for this figure.

Article Snippet: The following chemicals were used in the different experiments: p38 inhibitor at 10 μM (Selleckchem SB302580) to allow for continuous proliferation of RPE‐1 cells following centrosome amplification ; nocodazole, 100 ng/ml (Sigma‐Aldrich); MG132, 10 μM (Sigma‐Aldrich) HSET inhibitor CW069 at 150 μM with DLD‐1 and 25 μM with RPE‐1 cells (Selleckchem, S7336); dynein inhibitor ciliobrevin D, at 50 μM with DLD‐1 and 20 μM with RPE‐1 cells (Calbiochem, 250401); APC/C inhibitors ProTAME 10 μM (BostonBiochem, I‐440) and APCIN 40 μM (BostonBiochem, I‐444); doxycycline, 1 μg/ml (Sigma‐Aldrich, D9891); and auxin, 500 μM (Sigma‐Aldrich, I5148).

Techniques: Inhibition, Immunofluorescence, Control, Western Blot

APC/C inhibition reduces DLBCL and MCL viability and induces caspase-3-mediated apoptosis. a , b Effect of proTAME treatment on cell viability. MCL and DLBCL cell lines were treated for 24 h with proTAME (3, 6 and 12 µM) and the effect on viability was determined using a CellTiter-Glo assay ( a ). The results are shown as % viability relative to control. The IC-50 values for each cell line were calculated using Prism ( b ). c , d Effect of proTAME treatment on apoptosis. The effect of proTAME treatment on apoptosis ( c ) and percentage of active caspase-3- positive cells ( d ) was determined after 24 h, using an Annexin V/7′-AAD staining, followed by flow-cytometric analysis, and active caspase-3 staining followed by flow-cytometric analysis. The percentage of apoptotic cells are the sum of the percentage of Annexin V and Annexin V/7′-AAD positive cells. The results shown in graphs a , c and d are the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001. e , f The effect of proTAME treatment on primary patient samples. The viability of the proTAME-treated MCL ( e ) and DLBCL ( f ) patient samples was determined after 24 h. The results are shown as % viability relative to control

Journal: British Journal of Cancer

Article Title: The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

doi: 10.1038/s41416-019-0471-0

Figure Lengend Snippet: APC/C inhibition reduces DLBCL and MCL viability and induces caspase-3-mediated apoptosis. a , b Effect of proTAME treatment on cell viability. MCL and DLBCL cell lines were treated for 24 h with proTAME (3, 6 and 12 µM) and the effect on viability was determined using a CellTiter-Glo assay ( a ). The results are shown as % viability relative to control. The IC-50 values for each cell line were calculated using Prism ( b ). c , d Effect of proTAME treatment on apoptosis. The effect of proTAME treatment on apoptosis ( c ) and percentage of active caspase-3- positive cells ( d ) was determined after 24 h, using an Annexin V/7′-AAD staining, followed by flow-cytometric analysis, and active caspase-3 staining followed by flow-cytometric analysis. The percentage of apoptotic cells are the sum of the percentage of Annexin V and Annexin V/7′-AAD positive cells. The results shown in graphs a , c and d are the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001. e , f The effect of proTAME treatment on primary patient samples. The viability of the proTAME-treated MCL ( e ) and DLBCL ( f ) patient samples was determined after 24 h. The results are shown as % viability relative to control

Article Snippet: The APC/C inhibitor proTAME was obtained from R&D Systems (Oxon, UK).

Techniques: Inhibition, Glo Assay, Control, Staining

Pharmacological inhibition of the APC/C with proTAME results in a metaphase arrest. a Effect of proTAME treatment on the substrates of the APC/C co-activators Cdc20 and Cdh1. Expression of cyclin B1 and Skp2 was determined at different timepoints in the MCL and DLBCL cell lines after synchronisation and release into proTAME treatment (3 µM in SU-DHL-6 and 6 µM in Jeko-1 and U2932). APC3 and β-actin were used as indicators of mitotic arrest and loading control. Arrows show the phosphorylated APC3. One experiment representative of three is shown (the results shown are from the same experiment as those shown in Fig. ). Normalisation was performed with Image J and quantification relative to the M condition is shown. Bars represent the mean± SD of three independent experiments. b , c Effect of proTAME treatment on cell cycle progression. The effect on cell cycle progression was determined using PI staining on cell lines treated with 3 µM (SU-DHL-6) or 6 µM (Jeko-1 and U2932) proTAME for the indicated timepoints after synchronisation ( b ). To further examine the effect on mitosis, cytospins of these treated cells were stained with May–Grünwald Giemsa. For each sample, 3 × 100 cells were counted per cytospin. Quantification of the percentage of cells in the metaphase is shown ( c ). The results shown in each graph are the mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01. M = mitosis, −4 = 4 h before mitosis, −2 = 2 h before mitosis, +3 = 3 h after mitosis and +6 = 6 h after mitosis

Journal: British Journal of Cancer

Article Title: The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

doi: 10.1038/s41416-019-0471-0

Figure Lengend Snippet: Pharmacological inhibition of the APC/C with proTAME results in a metaphase arrest. a Effect of proTAME treatment on the substrates of the APC/C co-activators Cdc20 and Cdh1. Expression of cyclin B1 and Skp2 was determined at different timepoints in the MCL and DLBCL cell lines after synchronisation and release into proTAME treatment (3 µM in SU-DHL-6 and 6 µM in Jeko-1 and U2932). APC3 and β-actin were used as indicators of mitotic arrest and loading control. Arrows show the phosphorylated APC3. One experiment representative of three is shown (the results shown are from the same experiment as those shown in Fig. ). Normalisation was performed with Image J and quantification relative to the M condition is shown. Bars represent the mean± SD of three independent experiments. b , c Effect of proTAME treatment on cell cycle progression. The effect on cell cycle progression was determined using PI staining on cell lines treated with 3 µM (SU-DHL-6) or 6 µM (Jeko-1 and U2932) proTAME for the indicated timepoints after synchronisation ( b ). To further examine the effect on mitosis, cytospins of these treated cells were stained with May–Grünwald Giemsa. For each sample, 3 × 100 cells were counted per cytospin. Quantification of the percentage of cells in the metaphase is shown ( c ). The results shown in each graph are the mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01. M = mitosis, −4 = 4 h before mitosis, −2 = 2 h before mitosis, +3 = 3 h after mitosis and +6 = 6 h after mitosis

Article Snippet: The APC/C inhibitor proTAME was obtained from R&D Systems (Oxon, UK).

Techniques: Inhibition, Expressing, Control, Staining

proTAME inactivates the anti-apoptotic proteins Bcl-2 and Bcl-xL and sensitises MCL and DLBCL cells to venetoclax. a Effect of proTAME treatment on the expression and phosphorylation of Bcl-2, Bcl-xL and Mcl-1. The expression of Mcl-1, Cdc4, Bcl-2 and Bcl-xL protein and phosphorylation of Bcl-2 and Bcl-xL was determined at different timepoints in the synchronised MCL and DLBCL cell lines after proTAME treatment (3 µM in SU-DHL-6 and 6 µM in Jeko-1 and U2932) using western blot. APC3 and β-actin were used as indicators of mitotic arrest and loading control. Arrows show the phosphorylated APC3. One experiment representative of three is shown (the results shown are from the same experiment as those shown in Fig. ). Normalisation was performed with Image J and quantification relative to the M condition is shown. Bars represent the mean± SD of three independent experiments. M = mitosis, −4 = 4 h before mitosis, −2 = 2 h before mitosis, +3 = 3 h after mitosis and +6 = 6 h after mitosis, ND = not detected. b The anti-lymphoma effect of APC/C targeting in combination with the BH3 mimetic ABT-199. Apoptosis of proTAME (pT) and ABT-199-treated Jeko-1, SU-DHL-6 and U2932 cells was determined after 48 h using Annexin V/7′-AAD staining followed by flow-cytometric analysis. The sum of the percentage of Annexin V and Annexin V/7′-AAD-positive cells are shown. The results shown in each graph are the mean ± SD of four independent experiments. Each combination was compared with both single agents. ** p < 0.01 and *** p < 0.001

Journal: British Journal of Cancer

Article Title: The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

doi: 10.1038/s41416-019-0471-0

Figure Lengend Snippet: proTAME inactivates the anti-apoptotic proteins Bcl-2 and Bcl-xL and sensitises MCL and DLBCL cells to venetoclax. a Effect of proTAME treatment on the expression and phosphorylation of Bcl-2, Bcl-xL and Mcl-1. The expression of Mcl-1, Cdc4, Bcl-2 and Bcl-xL protein and phosphorylation of Bcl-2 and Bcl-xL was determined at different timepoints in the synchronised MCL and DLBCL cell lines after proTAME treatment (3 µM in SU-DHL-6 and 6 µM in Jeko-1 and U2932) using western blot. APC3 and β-actin were used as indicators of mitotic arrest and loading control. Arrows show the phosphorylated APC3. One experiment representative of three is shown (the results shown are from the same experiment as those shown in Fig. ). Normalisation was performed with Image J and quantification relative to the M condition is shown. Bars represent the mean± SD of three independent experiments. M = mitosis, −4 = 4 h before mitosis, −2 = 2 h before mitosis, +3 = 3 h after mitosis and +6 = 6 h after mitosis, ND = not detected. b The anti-lymphoma effect of APC/C targeting in combination with the BH3 mimetic ABT-199. Apoptosis of proTAME (pT) and ABT-199-treated Jeko-1, SU-DHL-6 and U2932 cells was determined after 48 h using Annexin V/7′-AAD staining followed by flow-cytometric analysis. The sum of the percentage of Annexin V and Annexin V/7′-AAD-positive cells are shown. The results shown in each graph are the mean ± SD of four independent experiments. Each combination was compared with both single agents. ** p < 0.01 and *** p < 0.001

Article Snippet: The APC/C inhibitor proTAME was obtained from R&D Systems (Oxon, UK).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Staining

APC/C inhibition enhances the anti-lymphoma activity of the Cdc20/Cdh1 inhibitor apcin and the clinically relevant agent doxorubicin. a–c The anti-lymphoma effect of APC/C targeting in combination with apcin, doxorubicin or rituximab. Apoptosis of proTAME (pT) and/or apcin ( a ), doxorubicin ( b ) or rituximab ( c ) treated with Jeko-1, SU-DHL-6 and U2932 cells was determined after 48 h using Annexin V/7′-AAD staining followed by flow-cytometric analysis. The sum of the percentage of Annexin V and Annexin V/7′-AAD-positive cells are shown. The results shown in each graph are the mean ± SD of four independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 when compared with both single agents. Combination index (CI) values were calculated for the different drug concentrations by the Chou and Thalalay method using CompuSyn 1.0 software (nd: not determined, CI ≤ 1: synergistic)

Journal: British Journal of Cancer

Article Title: The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

doi: 10.1038/s41416-019-0471-0

Figure Lengend Snippet: APC/C inhibition enhances the anti-lymphoma activity of the Cdc20/Cdh1 inhibitor apcin and the clinically relevant agent doxorubicin. a–c The anti-lymphoma effect of APC/C targeting in combination with apcin, doxorubicin or rituximab. Apoptosis of proTAME (pT) and/or apcin ( a ), doxorubicin ( b ) or rituximab ( c ) treated with Jeko-1, SU-DHL-6 and U2932 cells was determined after 48 h using Annexin V/7′-AAD staining followed by flow-cytometric analysis. The sum of the percentage of Annexin V and Annexin V/7′-AAD-positive cells are shown. The results shown in each graph are the mean ± SD of four independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 when compared with both single agents. Combination index (CI) values were calculated for the different drug concentrations by the Chou and Thalalay method using CompuSyn 1.0 software (nd: not determined, CI ≤ 1: synergistic)

Article Snippet: The APC/C inhibitor proTAME was obtained from R&D Systems (Oxon, UK).

Techniques: Inhibition, Activity Assay, Staining, Software

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apc c inhibitor protame (MedChemExpress)

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1) Product Images from "Targeting the splicing factor CWC22 induces mitotic slippage through repression of BubR1 expression and CDK1 activity in cancer cells"

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