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: Annals of Hematology

Article Title: Inhibition of CDC20 suppresses the development and progression of mantle cell lymphoma through PI3K/AKT pathway

doi: 10.1007/s00277-026-06926-0

Figure Lengend Snippet: Apcin could inhibit MCL cell proliferation, migration and invasion, and induce cell apoptosis and cell cycle arrest. ( A ) Relative CDC20 mRNA expression after 24 h apcin treatment and relative CDC20 protein expression after 24 h and 48 h apcin treatment in Z138, Mino and Rec1. ( B ) MCL cell lines Z138, Mino and Rec1 were exposed to apcin (50, 100, 200 µM) for 24, 48, and 72 h. Cell viability was assessed using CCK-8 assay and calculated as (OD450 of treatment group/OD450 of control group) × 100%. The IC50 values of apcin at 72 h in Z138, Mino, and Rec1 was calculated by GraphPad Prism. ( C ) Z138, Mino and Rec1 treated with 50 µM or 100 µM apcin for 48 h were analyzed for proliferation using EdU incorporation assay via flow cytometry. ( D ) Cell apoptosis was quantified by Annexin V-FITC/PI staining after 48 h apcin treatment. Total apoptosis rate represented the sum of early (lower right quadrant) and late apoptosis (upper right quadrant) rate. Compared with their respective control groups, the total apoptosis rate increased by 21.47% (50 µM) and 51.98% (100 µM) in Z138; by 8.78% (50 µM) and 22.91% (100 µM) in Mino; and by 6.65% (50 µM) and 45.04% (100 µM) in Rec1. ( E ) MMP was evaluated using JC-1 probe in Z138 and Mino cells following 48 h apcin treatment. Results are expressed as red/green fluorescence intensity ratio. ( F ) WB analysis of apoptosis-related proteins after 24 h and 48 h apcin treatment. ( G ) Cell cycle distribution was analyzed by PI staining after 48 h apcin exposure. ( H and I ) Transwell migration ( H ) and invasion ( I ) assays were conducted following 48 h apcin treatment. Images were captured using inverted microscopy. ( J ) WB assessed migration/invasion-related protein expression after 24 h and 48 h apcin treatment. All data were presented as mean ± SD from ≥ 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, ns meant no significance

Article Snippet: WB analysis was conducted using anti-CDC20 antibody (1:1000, sc-13162, Santa Cruz, USA), with β-actin (1:15000, 66009-1-Ig, Proteintech, China) as a loading control.

Techniques: Migration, Expressing, CCK-8 Assay, Control, Flow Cytometry, Staining, Fluorescence, Inverted Microscopy

Journal: Annals of Hematology

Article Title: Inhibition of CDC20 suppresses the development and progression of mantle cell lymphoma through PI3K/AKT pathway

doi: 10.1007/s00277-026-06926-0

Figure Lengend Snippet: CDC20 knockdown suppressed cell proliferation, migration and invasion, and promoted apoptosis and cell cycle arrest in Z138 cells. ( A ) Relative expression of CDC20 mRNA and CDC20 protein in Z138-shNC cells and Z138-shCDC20 cells. ( B ) Cell viability was assessed by CCK-8 assay at 0, 24, and 48 h, and presented as OD450 values. ( C ) Apoptosis was measured after culture for 48 h based on 7-AAD/PE staining. Total apoptosis meant combined early (lower right quadrant) and late apoptosis (upper right quadrant) populations. WB analysis of apoptosis-related proteins was performed in parallel. ( D ) Cell cycle distribution was analyzed by PI staining after culture for 48 h. ( E ) Migration and invasion abilities were evaluated by Transwell assay after culture for 48 h. Images were captured using inverted microscopy. WB analysis of migration/invasion-related proteins was conducted. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: WB analysis was conducted using anti-CDC20 antibody (1:1000, sc-13162, Santa Cruz, USA), with β-actin (1:15000, 66009-1-Ig, Proteintech, China) as a loading control.

Techniques: Knockdown, Migration, Expressing, CCK-8 Assay, Staining, Transwell Assay, Inverted Microscopy

Journal: Annals of Hematology

Article Title: Inhibition of CDC20 suppresses the development and progression of mantle cell lymphoma through PI3K/AKT pathway

doi: 10.1007/s00277-026-06926-0

Figure Lengend Snippet: Apcin demonstrated anti-tumor efficacy and safety in the Z138 xenograft model. ( A ) Tumor growth curves of the control group and the apcin group. ( B ) Comparison of tumor weight between the two groups. ( C ) Comparison of spleen weight between the two groups. ( D and E ) Representative photographs of harvested tumors ( D ) and spleens ( E ) from both groups. ( F ) Body weight monitoring of mice in the control group and the apcin group. ( G ) Representative HE images (×200 magnification; scale bar, 100 μm) of heart, liver, and kidney tissues of mice in both groups. ( H ) Comparison of relevant parameters in blood biochemistry of mice between the two groups. ( I ) Comparison of relevant parameters in blood routine of mice between the two groups. ( J ) Representative IHC images of CDC20, cleaved PARP and Ki67 staining of tumor tissues in the control group and the apcin group (×200 magnification; scale bar, 100 μm). ( K ) Quantification analysis of CDC20, cleaved PARP and Ki67 expression in the control group and the apcin group calculated by MOD values. ( L ) WB validation of CDC20 and cleaved PARP expression in both groups. ** P < 0.01, *** P < 0.001, ns meant no significance

Article Snippet: WB analysis was conducted using anti-CDC20 antibody (1:1000, sc-13162, Santa Cruz, USA), with β-actin (1:15000, 66009-1-Ig, Proteintech, China) as a loading control.

Techniques: Control, Comparison, Staining, Expressing, Biomarker Discovery

Journal: Annals of Hematology

Article Title: Inhibition of CDC20 suppresses the development and progression of mantle cell lymphoma through PI3K/AKT pathway

doi: 10.1007/s00277-026-06926-0

Figure Lengend Snippet: CDC20 inhibition modulated PI3K/AKT pathway phosphorylation. ( A ) RNA-seq analysis of Z138-shNC cells and Z138-shCDC20 cells revealed that differential genes were enriched in the PI3K/AKT signaling pathway. ( B ) Both apcin and CDC20-knockdown treatment resulted in a significant reduction of p-PI3K and p-AKT protein expression. *** P < 0.001

Article Snippet: WB analysis was conducted using anti-CDC20 antibody (1:1000, sc-13162, Santa Cruz, USA), with β-actin (1:15000, 66009-1-Ig, Proteintech, China) as a loading control.

Techniques: Inhibition, Phospho-proteomics, RNA Sequencing, Knockdown, Expressing