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: Targeting mGlyR with nanobodies for depression

doi: 10.1038/s41467-026-68339-x

Figure Lengend Snippet: a Schematic of the nanobody development pipeline. A phage library was constructed from leukocytes of a llama immunized with mGlyR, followed by 3 rounds of panning on mGlyR to enrich for specific binders, which were isolated and tested. b Schematic of the detection strategy in flow cytometry experiments. c Analysis of nanobody binding by flow cytometry of HEK cells transiently expressing mGlyR incubated with or without Nb20 and anti-myc-APC conjugated antibody. Percentages of cells in each quadrant are indicated. d Dose-response profiles of representative Nb20 binding experiment to cells expressing mGlyR in flow cytometry experiments. Concentrations of Nb20 are shown. e Quantification of data in panel D. Error bars are SEM values ( n = 3 independent experiments). f Schematic of the surface plasmon resonance (SPR) assays that detect Nb20 binding to the chip containing immobilized recombinant ectodomain of mGlyR (Ecto-mGlyR). g SPR sensorgram of binding and dissociation of Nb20.

Article Snippet: Nanobody-20 (Nb20) and 10 μl of anti-myc-APC conjugated antibody (R&d Systems #IC3696A) were added and incubated in the dark with rotation, at 4 °C for 1 h. After 3 washes, cells were analyzed in the LSR-II BD flow cytometer.

Techniques: Construct, Isolation, Flow Cytometry, Binding Assay, Expressing, Incubation, SPR Assay, Recombinant