Journal: Nature Communications

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1038/s41467-026-70437-9

Figure Lengend Snippet: UBA5 knock-down JEG-3 cells (shUBA5) or control cells (shNT) were infected with ZIKV H/PF/2013 wild-type strain (a-c) or ZIKV H/PF/2013 expressing Renilla luciferase (d) (MOI = 0.1). At 24 hpi, abundance of intracellular viral proteins ( a ) and viral RNA ( b ) were determined by western blotting and RT-qPCR ( b ), respectively. Infectious particle production was determined by PFU assay on cell culture supernatants ( c ), while viral replication was determined by luciferase activity ( d ). e Schematic representation of the UFMylation pathway and its main players. f Western blot analysis of UFMylation pathway activity after the complementation of UBA5 knock-out (KO) JEG-3 cells with wild-type (WT) or UFMylation-null UBA5 mutants. g Kinetics of infectious particle release in wild-type (WT) or UBA5 KO JEG-3 cells. Cells were infected with ZIKV H/PF/2013 (MOI = 0.01), supernatants collected at different times post-infection and analysed by PFU assay. h UBA5 KO JEG-3 cells complemented with either wild-type (WT) or UFMylation-null UBA5 mutants (L397R, Mut1; and M401R, Mut2) , were infected with ZIKV H/PF/2013 (MOI = 0.01) and virus titers measured in the supernatants at 48 hpi by PFU assay. b – d , g and h , n = 3 biological replicates; bars (or circles in g) represent the mean and error bars represent the standard deviation of the mean. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test ( b , c ), two-tailed unpaired t-test on log 10 -transformed PFU/mL values with Welch’s correction and Holm–Šidák correction for multiple comparisons ( g ) or one-way ANOVA with Dunnett’s multiple comparisons test ( h ). a , f representative immunoblots from n = 3 biological replicates are shown.

Article Snippet: The rabbit polyclonal anti-human UBA5 (12093-1-AP, WB: 1:1’000), HSD17B7 (14854-1-AP; WB, 1:1’000), HACD3 (28572-1-AP; WB, 1:1’000), mouse anti-Calnexin (66903-1-Ig; WB, 1:1’000; IF, 1:500), ChromoTek monoclonal rat anti-HA (7c9; 1:500) antibodies, and secondary anti-rat horseradish peroxidase (HRP)-conjugated antibodies (SA00001-15; WB, 1:1’000), were purchased from Proteintech.

Techniques: Knockdown, Control, Infection, Expressing, Luciferase, Western Blot, Quantitative RT-PCR, Cell Culture, Activity Assay, Knock-Out, Virus, Standard Deviation, Two Tailed Test, Transformation Assay

Journal: Nature Communications

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1038/s41467-026-70437-9

Figure Lengend Snippet: a Mock- or ZIKV-infected JEG-3 cells (ZIKV H/PF/2013, MOI = 0.1) were harvested at 24 and 48 hpi and subjected to non-reducing SDS-PAGE and western-blotting using antibodies against UFM1, NS4A and β-actin (representative of n = 2 independent experiments). b Immunoblot analysis of anti-FLAG immunoprecipitated extracts (right panels) and inputs (left panels) from mock- or ZIKV-infected (ZIKV H/PF/2013, MOI = 0.01) JEG-3 cells stably expressing FLAG UFM1∆SC. Eluates were stained with antibodies specific to UFMylation pathway members as indicated on the left (representative of n = 2 independent experiments) c Subcellular distribution of UFMylation pathway members upon ZIKV infection (UFSP2 and UFL1). JEG-3 cells infected with a ZIKV infectious molecular clone carrying an internal HA tag within NS4B (ZIKV-NS4B HA , MOI = 5) were fixed 24 hpi and stained with anti-UFSP2 (top two rows) or anti-UFL1 (bottom two rows), anti-HA and anti-NS3 antibodies. Scale bar = 10 μm. A representative experiment of n = 3 is shown. d Immunoblot analysis of anti-FLAG immunoprecipitated extracts (right panels) and inputs (left panels) from mock- and ZIKV-infected JEG-3 cells (ZIKV H/PF/2013, MOI = 0.01) stably expressing FLAG UFM1∆SC. Eluates were stained with antibodies specific to viral proteins as indicated on the left (representative of n = 2 independent experiments). Asterisk denote signal from prior anti-NS2B incubation. e Heat map of all detected UFMylation pathway members across the extended NS4B PPI networks. Intensity-based absolute quantification (iBAQ) of protein abundance of UFMylation-related host proteins across baits. N.I. not identified. f Replication of multiple orthoflaviviruses is inhibited in UBA5 KO cells. Control (gNT) or UBA5 KO (gUBA5) JEG-3 cells were infected with ZIKV, DENV2, JEV, WNV and YFV. At 48 hpi, infectious titers in the supernatant were determined by PFU assay. Herpes simplex virus 1 (HSV-1) was used as control. Bars represent the mean and error bars represent the standard deviation of the mean ( n = 3 biological replicates). Statistical significance was determined by two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: The rabbit polyclonal anti-human UBA5 (12093-1-AP, WB: 1:1’000), HSD17B7 (14854-1-AP; WB, 1:1’000), HACD3 (28572-1-AP; WB, 1:1’000), mouse anti-Calnexin (66903-1-Ig; WB, 1:1’000; IF, 1:500), ChromoTek monoclonal rat anti-HA (7c9; 1:500) antibodies, and secondary anti-rat horseradish peroxidase (HRP)-conjugated antibodies (SA00001-15; WB, 1:1’000), were purchased from Proteintech.

Techniques: Infection, SDS Page, Western Blot, Immunoprecipitation, Stable Transfection, Expressing, Staining, Incubation, Quantitative Proteomics, Control, Virus, Standard Deviation

Journal: Nature Communications

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1038/s41467-026-70437-9

Figure Lengend Snippet: a Schematic representation of DKM 2-93 inhibitory activity on UFMylation. DKM 2-93 competitively binds to the catalytic cysteine of UBA5, and prevents the activation of UFM1. b Dose-response curve of DKM 2-93 ZIKV antiviral activity in JEG-3 cells. Cells were treated with increasing concentrations of the inhibitor for 24 h, then infected with a Renilla luciferase ZIKV H/PF/2013 reporter virus (MOI = 0.1), and treated with the inhibitor for another 24 h. Cell viability and virus replication were determined at 24 hpi by resazurin and luciferase assays, respectively. c Western blot analysis of ZIKV-infected cells upon DKM 2-93 treatment, stained with anti-NS1 and anti-GAPDH specific antibodies (representative of n = 3 biological replicates). d Antiviral activity of DKM 2-93. JEG-3 cells were infected with ZIKV (ZIKV H/PF/2013, MOI = 0.01), and treated with either DMSO or two concentrations of DKM 2-93. Virus titers were determined by PFU assay 48 h.p.i. e Time-of-addition analysis of DKM 2-93 antiviral activity. JEG-3 cells were treated 3 h before (pre-; grey bar), during (co-; blue bar) or or 3 h after (post-; orange bar) virus absorption (ZIKV H/PF/2013, MOI = 0.01) with DKM 2-93 (32 µM). Virus titers were determined by PFU assay 48 h.p.i. f , g Huh7 cells were electroporated with in vitro transcribed RNA of a ZIKV subgenomic replicon expressing Renilla luciferase (sgZIKV-R2A), and treated with DKM 2-93 (32 µM) immediately thereafter. Luciferase activity was measured 4 h post electroporation to assess effects on viral RNA translation ( f ), and up to 96 h post electroporation to assess effects on viral RNA replication ( g ). b , d – g , n = 3 ( c , f ), n = 4 ( d , g ), and n = 5 ( e ) biological replicates; bars (circles for b and g) represent the mean and error bars represent the standard deviation of the mean. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test ( d ), two-way ANOVA with Tukey’s multiple comparisons test ( e ); two-way ANOVA with Sidak’s multiple comparisons test ( f ) or multiple unpaired two-tailed t-test on log 10 -transformed PFU/mL values with Welch correction and Holm–Šidák correction for multiple comparisons ( g ).

Article Snippet: The rabbit polyclonal anti-human UBA5 (12093-1-AP, WB: 1:1’000), HSD17B7 (14854-1-AP; WB, 1:1’000), HACD3 (28572-1-AP; WB, 1:1’000), mouse anti-Calnexin (66903-1-Ig; WB, 1:1’000; IF, 1:500), ChromoTek monoclonal rat anti-HA (7c9; 1:500) antibodies, and secondary anti-rat horseradish peroxidase (HRP)-conjugated antibodies (SA00001-15; WB, 1:1’000), were purchased from Proteintech.

Techniques: Activity Assay, Activation Assay, Infection, Luciferase, Virus, Western Blot, Staining, In Vitro, Expressing, Electroporation, Standard Deviation, Two Tailed Test, Transformation Assay

Journal: Nature Communications

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1038/s41467-026-70437-9

Figure Lengend Snippet: a Control (gNT) and UBA5 KO (gUBA5) JEG-3 cells cultured for two days were imaged by confocal microscopy. Representative images of mitochondria labeled with anti-COXIV (green) and the nuclear stain DAPI (blue). Scale bar, 20 µm. Quantitative analysis of mitochondrial morphology using the Mitochondrial Analyzer plugin in ImageJ/Fiji. b – g represent the mean area which calculates the average size of individual mitochondria per cell ( b ), the mean perimeter ( c ), the mean form factor (perimeter² / 4π × area) which reflects the mitochondrial shape, with higher values indicating more elongated structures and values closer to 1 indicating rounder mitochondria ( d ), the aspect ratio, the ratio of the major axis to the minor axis with higher AR > 1.5– 2.0 indicates elongated mitochondria associated with fusion ( e ), the mean branch length, the average length of individual mitochondrial branches within the network ( f ), and the branches per mitochondria ( g ) in UBA5 KO JEG-3 cells. h–k Knock-out of UBA5 impairs mitochondrial respiration. The Oxygen Consumption Rate (OCR) of UBA5 knock-out JEG-3 cells was measured at the indicated time points using the Seahorse technology. OCR values were first normalized to total protein content (µg per condition) and then to the mean basal OCR of control cells in each independent experiment ( h ). The basal respiration ( i ), ATP production ( j ), and maximal respiration ( k ) were quantified from the mitochondrial respiration profile. l–o ZIKV infection modulates mitochondrial respiration. Data from n = 3 biological replicates are shown in all panels. b – g floating bars (min. to max.), the middle line of the corresponds to the mean; h , l each circle represents the mean and error bars represent the standard deviation of the mean; and panels i – k , and m – o , the bars represent the mean and error bars represent the standard error of the mean. Statistical significance was determined by two-sided Mann-Whitney test ( b – g ) or unpaired two-sided t-test ( i – k , m – o ).

Article Snippet: The rabbit polyclonal anti-human UBA5 (12093-1-AP, WB: 1:1’000), HSD17B7 (14854-1-AP; WB, 1:1’000), HACD3 (28572-1-AP; WB, 1:1’000), mouse anti-Calnexin (66903-1-Ig; WB, 1:1’000; IF, 1:500), ChromoTek monoclonal rat anti-HA (7c9; 1:500) antibodies, and secondary anti-rat horseradish peroxidase (HRP)-conjugated antibodies (SA00001-15; WB, 1:1’000), were purchased from Proteintech.

Techniques: Control, Cell Culture, Confocal Microscopy, Labeling, Staining, Knock-Out, Infection, Standard Deviation, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: USP1 driven mitotic dysregulation and PLK1 stabilization confer Lenvatinib resistance in hepatocellular carcinoma

doi: 10.1186/s13046-026-03683-w

Figure Lengend Snippet: USP1 stabilizes PLK1 through deubiquitination. A Immunoblot analysis of PLK1 expression in HCC cells subjected to different treatments. B & C PLK1 expression in HCC cells treated with cycloheximide (CHX) at the indicated time points. Quantification of PLK1 levels was performed relative to β-actin. D & E Ubiquitination levels of PLK1 were analyzed by immunoprecipitation using an anti-PLK1 antibody, followed by immunoblotting with an anti-ubiquitin antibody. F PLK1 expression in HCC cells treated with CHX at indicated time points in the Control, WT, and C90S groups. G Immunoblot analysis of Flag-tagged USP1 and PLK1 expression in Huh7 and Hep3B cells transfected with Control, WT, and C90S plasmids. H Ubiquitination levels of PLK1 in Huh7 and Hep3B cells transfected with Control, WT, and C90S plasmids. I HCC cells treated with ML323 and transfected with HA-Ub, HA-K48-Ub, or HA-K63-Ub plasmids. Then PLK1 ubiquitination linkage was analyzed. J HCC cells transfected with HA-Ub or HA-K48-Ub in the presence of ShNC or ShUSP1-1/2. Cell lysates were analyzed by immunoblotting using anti-PLK1 and anti-USP1 antibodies

Article Snippet: Following blocking, the cells were incubated overnight at 4 °C with primary antibodies: anti-USP1 (Proteintech, Cat#1434-1-AP, 1:1000 v/v), anti-PLK1 (Cell Signaling, Cat#208G4, 1:500 v/v), anti-STUB1 (Santa Cruz, Cat#sc133066, 1:500 v/v).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Immunoprecipitation, Control, Transfection

Journal: Science Advances

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

doi: 10.1126/sciadv.aeb2345

Figure Lengend Snippet: ( A ) Representative images of USP8 localization in oocytes. Mouse oocytes were immunostained with USP8 and α-tubulin antibodies. Scale bar, 20 μm. ( B ) Representative images of USP8-mCherry localization during mouse oocyte meiosis. Mouse oocytes were microinjected with USP8-mCherry mRNA at the GV stage, maintained for 2 hours in 50 μM IBMX before being washed into IBMX-free medium to allow development to GVBD and MI stages, followed by DNA staining with Hoechst. Scale bar, 20 μm. ( C ) Protein levels of USP8 during oocyte meiosis were examined at the GV, GVBD, MI, and MII stages using immunoblotting analysis. The blots were probed with anti-USP8 antibody, anti–Cyclin B1 antibody, and anti-Cofilin antibody, respectively. The band intensity of USP8 was normalized with that of Cofilin. The GV-stage oocyte is characterized by a distinct GV, enabling clear morphological identification. The GVBD stage is defined by the recent breakdown and disappearance of the GV, whereas the MII stage can be identified by the presence of a prominent first polar body. Meanwhile, Cyclin B1 protein levels are dynamically regulated during oocyte meiotic maturation, gradually increasing from the GV to MI stage and subsequently decreasing at the MII stage compared to MI. Thus, these Cyclin B1 expression patterns can serve as supplementary indicators for determining oocyte meiotic maturation stages.

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: Staining, Western Blot, Expressing

Journal: Science Advances

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

doi: 10.1126/sciadv.aeb2345

Figure Lengend Snippet: ( A ) Representative images of the occurrence of GVBD in control and USP8-KD. Scale bar, 100 μm. ( B ) The incidences of GVBD were quantified in control ( n = 118) and USP8-KD ( n = 108) oocytes. ( C ) Representative images of PBE in control, USP8-KD, and USP8-rescued oocytes at the time point of 7 hours post-GVBD. Scale bar, 100 μm. ( D ) Quantitative analysis of PBE rates was shown in control ( n = 126), USP8-KD ( n = 122), and USP8-rescue ( n = 116) oocytes at consecutive time points of post-GVBD. ( E ) Protein levels of USP8 were assessed by immunoblots in control, USP8-KD, and USP8-rescue oocytes. The blots were probed with USP8 and Cofilin antibodies. ( F ) The proportion of oocytes overriding MI arrest following nocodazole treatment was recorded in control ( n = 101), USP8-KD ( n = 102), and USP8-rescue ( n = 99) oocytes. Oocytes injected with the indicated siRNA and/or mRNA were cultured with 400 nM nocodazole from 4 hours post-GVBD, and the PBE rate was scored at 10 hours post-GVBD. Data were presented as the mean value (means ± SEM) of at least three independent experiments. * P < 0.05; ** P < 0.01; n.s., not significant.

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: Control, Western Blot, Injection, Cell Culture

Journal: Science Advances

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

doi: 10.1126/sciadv.aeb2345

Figure Lengend Snippet: ( A ) Representative images of spindle morphologies and chromosome alignment in control and USP8-KD oocytes. At 6 hours post-GVBD, oocytes were fixed and immunostained for α-tubulin and DNA (PI). Scale bar, 20 μm. ( B ) The rates of aberrant spindle with misaligned chromosome were recorded in control ( n = 49) and USP8-KD ( n = 52) oocytes. Normal MI oocytes display a characteristic barrel-shaped spindle with properly aligned chromosomes at the equator; if the spindle does not exhibit this typical morphology or if there are “lagging” chromosomes, the oocytes are considered abnormal. ( C and D ) The spindle length and area were measured in control ( n = 16) and USP8-KD ( n = 16) oocytes at 6 hours post-GVBD. ( E ) Representative images of the width of the MI plate in control and USP8-KD oocytes. At 6 hours after GVBD, oocytes were fixed and immunostained for α-tubulin and DNA (PI). Scale bar, 20 μm. ( F ) The width of the MI plate was measured in control ( n = 15) and USP8-KD ( n = 15) oocytes. Data of (B) were presented as the mean percentage (means ± SEM) of at least three independent experiments. Data of [(C), (D), and (F)] were presented as or the mean value (means ± SD) of at least three independent experiments. ** P < 0.01; *** 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: Control

Journal: Science Advances

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

doi: 10.1126/sciadv.aeb2345

Figure Lengend Snippet: ( A ) Representative images of K-MT attachment in control, USP8-KD, and USP8-rescue oocytes. At 6 hours after GVBD, oocytes were incubated in M2 medium at 4°C for 10 min to induce the depolymerization of unstable microtubules and then immediately fixed and immunostained for α-tubulin, CREST, and DNA (Hoechst). White arrows indicate nonconnected kinetochores. Scale bar, 2.5 μm. ( B ) The number of unattached kinetochores was recorded in control ( n = 12), USP8-KD ( n = 12), and USP8-rescue ( n = 12) oocytes. ( C ) Representative images of euploid and aneuploid MII eggs. Chromosome spreading was performed to count the number of chromosomes in control, USP8-KD, and USP8-rescue oocytes at 10 hours after GVBD. The total number of univalents is indicated by the yellow square. Scale bar, 7 μm. ( D ) The rates of aneuploid eggs were recorded in control ( n = 30), USP8-KD ( n = 34), and USP8-rescue ( n = 29) oocytes. Data of (D) were presented as the mean percentage (means ± SEM) of at least three independent experiments. Data of (B) were presented as the mean value (means ± SD) 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: Control, Incubation

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: Science Advances

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

doi: 10.1126/sciadv.aeb2345

Figure Lengend Snippet: ( A ) Quantitative analysis of PBE rates were shown in control ( n = 121), USP8-KD ( n = 121), and BUB3-rescue ( n = 117) oocytes at consecutive time points of post-GVBD. ( B ) Representative images of spindle morphologies and chromosome alignment in control, USP8-KD, and BUB3-rescue oocytes. At 6 hours post-GVBD, oocytes were fixed and immunostained for α-tubulin and DNA (PI). Scale bar, 20 μm. ( C ) The rates of aberrant spindle with misaligned chromosome were recorded in control ( n = 49), USP8-KD ( n = 50), and BUB3-rescue ( n = 43) oocytes. ( D and E ) The spindle length and area were measured in control ( n = 15), USP8-KD ( n = 15), and BUB3-rescue ( n = 15) oocytes at 6 hours post-GVBD. ( F ) The width of the MI plate was measured in control ( n = 14), USP8-KD ( n = 14), and BUB3-rescue ( n = 14) oocytes. ( G ) Representative images of euploid and aneuploid MII eggs. Chromosome spreading was performed to count the number of chromosomes in control, USP8-KD, and BUB3-rescue oocytes at 10 hours after GVBD. The total number of univalents is indicated by the yellow square. Scale bar, 7 μm. ( H ) The rates of aneuploid eggs were recorded in control ( n = 30), USP8-KD ( n = 29), and BUB3-rescue ( n = 27) oocytes. Data were presented as the mean percentage (means ± SEM) of at least three independent experiments. * P < 0.05; ** P < 0.01; *** 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: Control

Journal: Science Advances

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

doi: 10.1126/sciadv.aeb2345

Figure Lengend Snippet: ( A ) mRNA levels of BUB3 were determined by reverse transcription quantitative polymerase chain reaction in control and USP8-KD oocytes. ( B ) Protein levels of BUB3 in control, USP8-KD, and USP8-KD + MG132 (25 μM) oocytes. The blots were probed with BUB3 and Cofilin antibodies. DMSO, dimethyl sulfoxide. ( C ) Immunoblotting analysis showing BUB3 protein levels in control oocytes and oocytes treated with the broad-spectrum DUB inhibitor PR-619 (10 μM). ( D ) Immunoblotting analysis of BUB3 protein levels in control and DUB-IN-2 (20 μM)–treated oocytes, where DUB-IN-2 is a specific inhibitor of USP8 deubiquitinating activity. ( E ) Localization of BUB3 at the prometaphase I stage in control and DUB-IN-2–treated oocytes. At 3 hours after GVBD, oocytes were fixed and immunostained for BUB3, CREST, and DNA (Hoechst). Scale bar, 10 μm. ( F ) The relative fluorescence intensities of BUB3 to CREST were measured in control ( n = 200, kinetochores) and DUB-IN-2–treated ( n = 200, kinetochores) oocytes. The signal intensity of BUB3 was normalized with that of CREST. *** P < 0.001. ( G ) USP8 reduces the ubiquitination level of BUB3. Mouse oocytes were injected with the respective cRNAs and maintained for an additional 4 hours in 200 μM IBMX to allow for protein translation. Target proteins were immunoprecipitated using anti-HA beads and analyzed by Western blotting with anti-MYC and anti-HA antibodies. Input oocyte lysates were immunoblotted with an anti-Flag antibody to assess USP8 expression. ( H ) Working model of the mechanism that USP8 governs SAC to ensure oocyte euploidy. In normal oocytes, USP8 maintains BUB3 protein levels through its deubiquitinating activity, thereby sustaining the SAC activity in oocytes and ultimately preserving oocyte ploidy. However, in the absence of USP8, BUB3 is targeted for ubiquitin-mediated proteasomal degradation, leading to SAC inactivation and the production of aneuploid eggs.

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: Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Western Blot, Activity Assay, Fluorescence, Ubiquitin Proteomics, Injection, Immunoprecipitation, Expressing, Preserving