mad2  (Bethyl)

Journal: Science Advances

Article Title: Deubiquitinase USP8 regulates the spindle assembly checkpoint in oocytes

doi: 10.1126/sciadv.aeb2345

Figure Lengend Snippet: ( A ) Protein levels of the MCC were assessed by immunoblots in control and USP8-depleted oocytes. The blots were probed with USP8, BUBR1, CDC20, BUB3, MAD2, and Cofilin antibodies. ( B ) Localization of BUB3 at the prometaphase I stage in control, USP8-KD, and USP8-rescue oocytes. At 3 hours after GVBD, oocytes were fixed and immunostained for BUB3, CREST, and DNA (Hoechst). Scale bar, 10 μm. ( C ) The relative fluorescence intensities of BUB3 to CREST were measured in control ( n = 200, kinetochores), USP8-KD ( n = 200, kinetochores), and USP8-rescue ( n = 200, kinetochores) oocytes. The signal intensity of BUB3 was normalized with that of CREST. ( D ) Protein levels of BUB3 were assessed by immunoblots in control, USP8-KD, and USP8-rescue oocytes. ( E ) Co-IP result showing the USP8 interaction with BUB3 in oocytes. Mouse oocytes were microinjected with USP8-Flag and BUB3-HA cRNA together or USP8-Flag cRNA alone, maintained for a further 4 hours in 200 μM IBMX to allow time for translation. Target proteins were immunoprecipitated using anti-Flag beads and subjected to Western blotting with Flag and HA antibodies. Input oocyte lysates were immunoblotted with anti-HA antibody to determine the expression of BUB3. ( F ) Co-IP result showing the BUB3 interaction with USP8 in oocytes. Mouse oocytes were microinjected with BUB3-HA and USP8-Flag cRNA together or BUB3-HA cRNA alone, maintained for a further 4 hours in 200 μM IBMX to allow time for translation. Target proteins were immunoprecipitated using anti-HA beads and subjected to Western blotting with HA and Flag antibodies. Input oocyte lysates were immunoblotted with anti-Flag antibody to determine the expression of USP8. Data were presented as the mean percentage (means ± SEM) of at least three independent experiments. *** P < 0.001.

Article Snippet: The blots were further blocked in TBST containing 5% low-fat dry milk for 1 hour at room temperature and then incubated with USP8 (1:500; Abclonal, A7031), USP8 (1:500; Proteintech, 67321-1-Ig), Cyclin B1 (1:1000; Cell Signaling Technology, 4135), BUB3 (1:1000; Abcam, ab133699), BUBR1 (1:1000; Abcam, Ab28193), MAD2 (1:1000; Proteintech, 10337-1-AP), CDC20 (1:500; Proteintech, 10252-1-AP), BUB1 (1:1000; Abcam, Ab195268 ), MPS1 (1:500; Proteintech, 10381-1-AP), Flag (1:1000; Sigma-Aldrich, F3165), HA (1:1000; Sigma-Aldrich, H9658), MYC (1:1000; Cell Signaling Technology, 2278), Normal Rabbit IgG (1:1000; Cell Signaling Technology, 2729), or Cofilin (1:5000; Proteintech, 66057-1-Ig) antibodies at 4°C overnight.

Techniques: Western Blot, Control, Fluorescence, Co-Immunoprecipitation Assay, Immunoprecipitation, Expressing

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Enhanced CDRs correlated with prostate cancer prognosis. (A) Visualization of core genes' dependency in different prostate cancer cells with CRISPR-Cas9 and siRNA screening. (B, C) Survival analysis of CDRs in different CRPC patient cohorts. (D) The relative expression of CDRs in normal prostate tissues and prostate cancer tissues in the TCGA-PRAD cohort. Gleason score and tumor stage correlation analysis of CDRs in different prostate cancer patient cohorts. All prostate patient cohorts were indicated in the corresponding panels. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2).

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: CRISPR, Expressing, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs linked with neuroendocrine features in prostate cancer cohorts. (A) The Venn diagram illustrates the genes specifically enhanced in prostate cancer compared with normal prostate tissues and NEPC compared with adenocarcinoma. (B, C) The relative expression of CDRs in NEPC compared with adenocarcinoma in different prostate cancer cohorts. (D) Correlation analysis of CDRs with NE score. The red indicates the higher NE score. (E, F) Pearson correlation analysis of CDRs with NE score CRPC cohort. (G) The heatmap indicates the relative expression of CDRs and AR, as well as KLK3, a transcription activity indication of AR. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). NEPC, neuroendocrine prostate cancer; NE, neuroendocrine; AR, androgen receptor; KLK3, kallikrein-related peptidase 3.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Expressing, Activity Assay, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs ablation suppressed prostate cancer cell proliferation. (A) The circuit diagram illustrates the correlation among CDRs co-expressed gene sets. (B) Venn analysis of the overlap of CDRs co-expressed genes. (C) The heatmap shows the relative expression of CDRs overlapped co-expressed genes in patients with NE signature high and low groups. (D – F) The heatmap illustrates the correlation of CDRs co-expressed genes with CDRs in ADPC (D) and CRPC (E, F) patient cohorts. (G, H) KEGG pathway analysis (G) and GSEA analysis (H) of enriched biological processes of CDRs and CDRs co-expressed genes. (I, J) The diagram illustrates the working model of CRISPR-Cas13 for RNA silencing and knockdown efficiency of CDRs with the corresponding gRNAs. (K, L) Cell growth assays indicate the impact of CDRs knockdown on the viability of different prostate cancer cells. (M) Western blotting analysis of the expression of cell cycle-regulated genes, including CCND1, CDK1, and p-CDK1, after CDRs knockdown. ∗∗ p < 0.01. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). NE, neuroendocrine; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; CCND1, cyclin D1; CDK1, cyclin-dependent kinase 1.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Expressing, CRISPR, Knockdown, Western Blot, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs were transcriptionally regulated by the RB1/E2F1 axis in prostate cancer. (A) The diagrams show the canonical E2F1 motif location within the promoter of CDRs. (B) ChIP-sequencing E2F1 enrichment peak within the promoter of CDRs. (C) The relative enrichment of E2F1 within the promoter of CDRs was determined using standard ChIP-qPCR. (D) ChIP-sequencing peaks show the relative enrichment of E2F1 in CDRs' promoters after RB1 is known. (E – G) The relative expression of CDRs in CRPC patients with different RB1 deletion status. (H) CDRs expression in patients with RB1 deletion mutations from different prostate cancer cohorts. (I) qRT-PCR detected the relative expression of CDRs after RB1 or E2F1 knockdown with CRISPR-Cas13. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). RB1, retinoblastoma tumor suppressor 1; E2F1, early 2 factor 1; ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time PCR.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: ChIP-sequencing, ChIP-qPCR, Expressing, Quantitative RT-PCR, Knockdown, CRISPR, Ubiquitin Proteomics, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Virtual screening identified compounds that suppressed advanced prostate cancer. (A) The diagram illustrates structure-based virtual screening strategies for CDRs-targeted compounds. (B) The heatmap shows the binding affinity of compounds with CDRs. The red indicated a higher binding affinity of compounds with the corresponding compounds. (C, D) Cell viability assays were used to determine the tumor suppressive effect of compounds with high CDRs-binding affinity in different prostate cancer cell models. (E – G) The 2D structure of Q199, XDD60, and A79, which exhibits the most significant anti-tumor efficacy in prostate cancer cell models. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2).

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Binding Assay, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Compounds targeting CDRs exhibited superior anti-tumor efficacy compared with AR antagonists. (A – E) The tumor cell growth inhibition effects of different dosages of Q199, XDD60, and A79, as well as enzalutamide, were determined with CCK-8 assays (A–D), and the IC 50 of each agent was calculated with three independent experiments (E). (F, G) The histograms show the relative cell viability after being treated with 5 M of Q199, XDD60, or A79 alone, or a combination. (H) The Venn diagram shows the overlap of Q199, XDD60, and A79 potential targets predicted with SwissTargetPrediction ( http://swisstargetprediction.ch/ ). Molecular docking shows the binding of CDRs with Q199, XDD60, and A79. (I) The lowest binding (LB) affinity of CDRs with Q199, XDD60, and A79. ns, not significant. ∗∗ p < 0.01. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). AR, androgen receptor.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Inhibition, CCK-8 Assay, Binding Assay, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Ribosome Quality Control Mitigates Proteotoxic Stress in Aneuploid Cells

doi: 10.64898/2026.01.19.700285

Figure Lengend Snippet: A. Representative immunoblot and replicate quantitation of processed-Keima levels in HCT116 RPS3-Keima cells treated for 24 hours with 125nM, 250nM or 500nM Mps1i or DMSO (Ctrl); vinculin was used as loading control. Mean ± SEM, n =4; Kruskal-Wallis test, followed by Dunn’s multiple comparison test: * indicates p =0.0150. B. Representative immunoblot and replicate quantitation of processed-Keima levels in HCT116 RPL28-Keima cells treated for 24 hours with 125nM, 250nM or 500nM Mps1i or DMSO (Ctrl); vinculin was used as loading control. Mean ± SEM, n =4; Kruskal-Wallis test, followed by Dunn’s multiple comparison test: * indicates p =0.0329 (250nM) or p =0.0329 (500nM). C. Representative immunoblots and replicate quantitation of the HCT116 RPS3-Keima cells upon MAD2 or BUB1 siRNA (or non-targeting siRNA); increased levels of processed-Keima indicates ribosome degradation; MAD2 or BUB1 were blotted as knock-down control. Vinculin and tubulin used as loading control. Mean ± SEM, n =4; one sample and Wilcoxon test (each siRNA vs respective non-targeting siRNA=1): * indicates p =0.0257. D. Representative immunoblots and replicate quantitation of the HCT116 RPL28-Keima cells upon MAD2 or BUB1 siRNA (or non-targeting siRNA); increased levels of processed-Keima indicates ribosome degradation; MAD2 or BUB1 have been blotted as knock-down control. Vinculin and tubulin been used as loading control. Mean ± SEM, n =4; one sample and Wilcoxon test (each siRNA vs respective non-targeting siRNA=1): * indicates p =0.0292. E. Representative live-cell images and replicate quantitation of indicated HCT116 Ribo-Keima cells lines treated with Mps1i pulse or DMSO (Ctrl) and collected at 24 hours. Increased Red-Keima intensity indicates ribosome degradation; Hoechst was used to stain DNA; scale bars, 50μm. Upper quartile, lower quartile and median of each violin plot are shown, n =12 fields of view; unpaired Student’s t-test: **** indicates p <0.0001. F. Representative images and replicate quantitation of RFP/GFP ratio in RPE1 RPS3-RFP-GPF cells treated with Mps1i pulse or DMSO (Ctrl) and collected at 24 or 72 hours. RFP/GFP ratio was calculated as explained in Methods and visualised with a rainbow lookup table (calibration bar shown); cyan masks indicate primary nuclei; scale bars 50μm. Upper quartile, lower quartile and median of each violin plot are shown; n=3 biological replicates (30 fields of view each); Kruskal-Wallis test, followed by Dunn’s multiple comparison test: *** indicates p =0.0002, **** indicates p <0.0001.

Article Snippet: The following primary antibodies were used: BUB1 (#ab54893, Abcam), EGFR (in-house polyclonal ab against human EGFR aa 1172–1186, kindly provided by Prof. Di Fiore), GAPDH (#sc-32233, Santa Cruz Biotechnology), HA (#MMS-101P, BioLegend), HSF1 (#4356S, Cell Signaling Technology), Hsp27 (#ADI-SPA-800-D, Enzo Life Science), Hsp70/72 (#ADI-SPA-810-D, Enzo Life Science), Hsp90 (#4877S, Cell Signaling Technology), Keima-red (#M182-3M, MBL), MAD2 (#A300-301A, Bethyl Laboratories), MET (#sc-162, Santa Cruz Biotechnology), mTOR (#2983S, Cell Signaling Technology), PDGFRβ (#3169S, Cell Signaling Technology), Puromycin clone 12D10 (#MABE343, Merck), Tubulin (#T9026, Sigma-Aldrich), Vinculin (#V9131, Sigma-Aldrich), ZNF598 (#ab241092, Abcam).

Techniques: Western Blot, Quantitation Assay, Control, Comparison, Knockdown, Staining

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days after doxycycline (Dox) addition (mean and SD, two-tailed Student’s t test, CENP-C WT cells: n = 6; CENP-C ΔM12BD cells: n = 6; ** p < 0.01). (B) Representative images of DAPI-stained K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Scale bar, 50 μm. (C) Population of normal interphase cells, mitotic cells, and cells with micronuclei in K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Error bars indicate SEM. n = 3 independent experiments; 200 cells from each cell line were quantified in each experiment. (D) The growth curve of K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. K562 WT or CENP-C ΔM12BD cells were treated with or without Dox ( KIF18A OFF or ON). The cell numbers were normalized to those at time 0 of each line. (E) Cell-cycle distribution of conditional knockout of KIF18A in K562 WT cells at each day after Dox addition, based on FACS analysis. (F) Cell-cycle distribution of conditional knockout of KIF18A in K562 CENP-C ΔM12BD cells at each day after Dox addition, based on FACS analysis. (G and H) Quantification of cells with misaligned chromosomes in K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. The experimental scheme is shown. Cells were stained with antibodies against MAD2 (red) to detect misaligned chromosomes and CENP-T (green) as a kinetochore marker. DNA was stained with DAPI (blue). Arrowheads show typical MAD2-positive unaligned chromosomes. Scale bar, 10 μm. The cells with MAD2-positive chromosomes were quantified (H) (mean and SEM, two-tailed Student’s t test; n = 5 independent experiments; n.s., non-significant; ** p < 0.01). (I) Numbers of MAD2 positive kinetochores in each cell in each condition (WT KIF18A ON; WT KIF18A OFF; CENP-C ΔM12BD KIF18A ON; CENP-C ΔM12BD KIF18A OFF) (Mean and SEM, n = 5 independent experiments).

Article Snippet: Mouse anti-human MAD2 , Santa Cruz Biotechnology , Cat#sc-65492; RRID: AB_831526.

Techniques: Knock-Out, Two Tailed Test, Staining, Marker