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: BMC Cancer

Article Title: A signature of correlated CDC20 and UBCH10 expression indicates poor prognosis in primary head and neck squamous cell carcinoma

doi: 10.1186/s12885-025-14773-x

Figure Lengend Snippet: Correlated overexpression of CDC20 - UBCH10 promotes oncogenic properties in HNSC cell line, SCC084. Cells were ectopically transfected with 1000 ng of control or pCDNA5-FLAG-Cdc20 plasmid. Cells was processed for ( A ) Western blot analyses using antibodies against Cdc20, UbcH10 and β-actin (left panel), and their densitometric analyses (right panel); (* p < 0.05, ** p < 0.01, unpaired Student’s t-test), B Clonogenic assay (left panel, representative image & right panel, quantification of colonies); (* p < 0.05, Welch’s t-test), C Wound healing assay (left panel, representative image; top right panel, western blot representative image & bottom right panel, quantification of wound healing) (* p < 0.05, ns; not significant, unpaired student’s t-test), D Matrigel invasion assay (left panel, representative image & right panel, quantification of cell invasion) (* p < 0.05, unpaired student’s t-test), and, E Cell viability (MTT) assay (* p < 0.05, ** p < 0.01, ns; not significant, two way anova, Sidak’s multiple comparison test). F Cells were stably transduced with control or CDC20 -shRNA specific lentiviral vector plasmid. Cells was processed for ( F ) Western blot analyses using antibodies against Cdc20, UbcH10 and β-actin (left panel), and their densitometric analyses (right panel), G Colony formation assay (left panel, representative image & right panel, quantification of colonies), H Matrigel invasion assay (left panel, representative image & right panel, quantification of cell invasion), and, I Cell viability (MTT) assay. For all experiments the statistically analysed data are indicated as mean ± SD of three independent experiments

Article Snippet: For overexpression analysis, cells were transiently transfected with 1 μg of plasmid and harvested after 48 h. Also, human p55 CDC shRNA ( CDC20 ) target specific lentiviral plasmid (Santa Cruz Biotechnology, Inc.) along with packaging and envelope plasmids (psPAX2 and pMD2.G) were transfected into cultured HEK293T cells at 60% confluency.

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, Western Blot, Clonogenic Assay, Wound Healing Assay, Invasion Assay, MTT Assay, Comparison, Stable Transfection, Transduction, shRNA, Colony Assay