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: ACS sensors

Article Title: Development of a Microfluidic Flow Cytometer with a Uniform Optical Field (Uni-μFCM) Enabling Quantitative Analysis of Single-Cell Proteins and Its Applications in Leukemia Gating, Tumor Classification, and Hierarchy of Cancer Stem Cells.

doi: 10.1021/acssensors.3c01060

Figure Lengend Snippet: Figure 5. Uni-μFCM was combined with gene knockout to measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies. (A) Scatter plots of XRCC3, Cdc20, and BubR1 from paired tumor cell lines of salivary adenoid cystic carcinoma with low (SACC-83) and high (SACC-LM) lung metastatic capabilities. (B) Scatter plots of single- cell expressions of XRCC3, Cdc20, and BubR1 from colon tumor cell lines HCT116 P53+/+ and its subtype HCT116 P53−/−with P53 deficiency.

Article Snippet: Fluorescence-labeled antibodies used for cell staining included antibodies of OCT4-Alexa 488 purchased from Thermo Fisher, USA, CD3-Alexa 488, XRCC3-Alexa 488, c-Myc-PE, CD79a-PE, Cdc20-PE, KLF4-PerCP, LAMP1-PerCP, and BubR1-PerCP purchased from NOVUS, USA.

Techniques: Gene Knockout

Journal: British Journal of Cancer

Article Title: The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

doi: 10.1038/s41416-019-0471-0

Figure Lengend Snippet: Cdc20 mRNA expression is increased in DLBCL and MCL patients and is associated with worse survival in DLBCL patients. a , b Cdc20 and Cdh1 mRNA expression in DLBCL. Cdc20 ( a ) and Cdh1 ( b ) gene expression levels of B-cell samples ( n = 33), patients with ABC–DLBCL ( n = 190), patients with GCB–DLBCL ( n = 212) and patients with unclassified DLBCL ( n = 67) were obtained from the publicly available microarray datasets GSE10846 and GSE56315. Mean expression±SD is shown in red. ** p < 0.01 and *** p < 0.001. c , d Cdc20 and Cdh1 mRNA expression in indolent and aggressive MCL. Cdc20 ( c ) and Cdh1 ( d ) gene expression levels of patients with indolent ( n = 7) and aggressive MCL ( n = 15) were obtained from the GSE16455 dataset. Mean expression ± SD is shown in red. * p < 0.05. e , f Prognostic value of Cdc20 and Cdh1 mRNA levels in terms of overall survival in DLBCL. The prognostic value of Cdc20 ( e ) and Cdh1 ( f ) was determined in DLBCL patients receiving R-CHOP treatment from the Lenz cohort ( n = 233) using Maxstat analysis (cut-off value used for Cdc20 is 11.679 and for Cdh1 8.319). Data were analysed through genomicscape ( http://genomicscape.com ). g – j Immunohistochemical analysis of Cdc20 expression in primary DLBCL patient samples. Immunohistochemical analysis of Cdc20 expression in a DLBCL patient. A ×4 and ×40 magnification is shown ( g ). Percentage of Cdc20-positive lymphoma cells was counted in 34 DLBCL patients and plotted against the estimated prognosis according to the R-IPI score of the patients ( h ). Percentage of Cdc20-positive cells was also plotted against the molecular subtype ( i ) and double-expresser status ( j ) in 27 DLBCL patients

Article Snippet: All samples were stained with Cdc20 antibody (ATLAS Antibodies AB, Bromma, Sweden) using an automated immunostainer Benchmark XT (Roche Ventana, Basel, Switzerland).

Techniques: Expressing, Gene Expression, Microarray, Immunohistochemical staining

Journal: British Journal of Cancer

Article Title: The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

doi: 10.1038/s41416-019-0471-0

Figure Lengend Snippet: COX univariate and multivariate analysis of overall survival in R-CHOP-treated DLBCL ( n = 233) including Cdc20 gene expression

Article Snippet: All samples were stained with Cdc20 antibody (ATLAS Antibodies AB, Bromma, Sweden) using an automated immunostainer Benchmark XT (Roche Ventana, Basel, Switzerland).

Techniques:

Journal: British Journal of Cancer

Article Title: The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

doi: 10.1038/s41416-019-0471-0

Figure Lengend Snippet: Pharmacological inhibition of the APC/C with proTAME results in a metaphase arrest. a Effect of proTAME treatment on the substrates of the APC/C co-activators Cdc20 and Cdh1. Expression of cyclin B1 and Skp2 was determined at different timepoints in the MCL and DLBCL cell lines after synchronisation and release into proTAME treatment (3 µM in SU-DHL-6 and 6 µM in Jeko-1 and U2932). APC3 and β-actin were used as indicators of mitotic arrest and loading control. Arrows show the phosphorylated APC3. One experiment representative of three is shown (the results shown are from the same experiment as those shown in Fig. ). Normalisation was performed with Image J and quantification relative to the M condition is shown. Bars represent the mean± SD of three independent experiments. b , c Effect of proTAME treatment on cell cycle progression. The effect on cell cycle progression was determined using PI staining on cell lines treated with 3 µM (SU-DHL-6) or 6 µM (Jeko-1 and U2932) proTAME for the indicated timepoints after synchronisation ( b ). To further examine the effect on mitosis, cytospins of these treated cells were stained with May–Grünwald Giemsa. For each sample, 3 × 100 cells were counted per cytospin. Quantification of the percentage of cells in the metaphase is shown ( c ). The results shown in each graph are the mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01. M = mitosis, −4 = 4 h before mitosis, −2 = 2 h before mitosis, +3 = 3 h after mitosis and +6 = 6 h after mitosis

Article Snippet: All samples were stained with Cdc20 antibody (ATLAS Antibodies AB, Bromma, Sweden) using an automated immunostainer Benchmark XT (Roche Ventana, Basel, Switzerland).

Techniques: Inhibition, Expressing, Control, Staining

Journal: British Journal of Cancer

Article Title: The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

doi: 10.1038/s41416-019-0471-0

Figure Lengend Snippet: APC/C inhibition enhances the anti-lymphoma activity of the Cdc20/Cdh1 inhibitor apcin and the clinically relevant agent doxorubicin. a–c The anti-lymphoma effect of APC/C targeting in combination with apcin, doxorubicin or rituximab. Apoptosis of proTAME (pT) and/or apcin ( a ), doxorubicin ( b ) or rituximab ( c ) treated with Jeko-1, SU-DHL-6 and U2932 cells was determined after 48 h using Annexin V/7′-AAD staining followed by flow-cytometric analysis. The sum of the percentage of Annexin V and Annexin V/7′-AAD-positive cells are shown. The results shown in each graph are the mean ± SD of four independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 when compared with both single agents. Combination index (CI) values were calculated for the different drug concentrations by the Chou and Thalalay method using CompuSyn 1.0 software (nd: not determined, CI ≤ 1: synergistic)

Article Snippet: All samples were stained with Cdc20 antibody (ATLAS Antibodies AB, Bromma, Sweden) using an automated immunostainer Benchmark XT (Roche Ventana, Basel, Switzerland).

Techniques: Inhibition, Activity Assay, Staining, Software