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: International journal of oncology

Article Title: CDC20 associated with cancer metastasis and novel mushroom‑derived CDC20 inhibitors with antimetastatic activity.

doi: 10.3892/ijo.2019.4791

Figure Lengend Snippet: Figure 1. CDC20 knockdown inhibits the migration of chemoresistant pancreatic cancer and metastatic breast cancer cells. PANC‑1 cells and MDA‑MB‑231 cells were transfected with scrambled siRNA or CDC20 siRNA. (A) After 48 h of transfection, western blot analysis of CDC20 in PANC‑1 cells was evaluated. (B) Migration of PANC‑1 cells in Transwell chambers. (C) After 48 h of transfection, western blot analysis of CDC20 in MDA‑MB‑231 cells was evaluated. (D) Migration of MDA‑MB‑231 cells in Transwell chambers. Each bar represents the mean ± standard deviation of three individual filters within one repre sentative experiment repeated at least twice. **P<0.05 vs. Control. CDC20, cell division cycle 20; siRNA, small interfering RNA.

Article Snippet: The PANC-1 or MDA-MB-231 cells were transfected with human CDC20 siRNA (cat. no. sc-156154) or control siRNA-A (cat. no. sc-37007) using siRNA Transfection Reagent (cat. no. sc-29528) from Santa Cruz Biotechnology, Inc. as previously described (27).

Techniques: Knockdown, Migration, Transfection, Western Blot, Standard Deviation, Control, Small Interfering RNA

Journal: International journal of oncology

Article Title: CDC20 associated with cancer metastasis and novel mushroom‑derived CDC20 inhibitors with antimetastatic activity.

doi: 10.3892/ijo.2019.4791

Figure Lengend Snippet: Figure 2. Overexpression of CDC20 promotes the migration of chemoresistant pancreatic cancer and breast cancer. PANC‑1 cells and MCF‑7 cells were transfected with FLAG‑tagged plasmid DNA with CDC20 or FLAG‑tagged plasmid DNA. (A) After 24 h of transfection, western blot analysis of CDC20 in PANC‑1 cells was performed. (B) Migration of PANC‑1 cells in Transwell chambers. (C) After 24 h of transfection, western blot analysis of CDC20 in MCF‑7 cells was performed. (D) Migration of MCF‑7 cells in Transwell chambers was determined. Each bar represents the mean ± standard deviation of three individual filters within one representative experiment repeated at least twice. **P<0.05 and ***P<0.001 vs. Control. CDC20, cell division cycle 20.

Article Snippet: The PANC-1 or MDA-MB-231 cells were transfected with human CDC20 siRNA (cat. no. sc-156154) or control siRNA-A (cat. no. sc-37007) using siRNA Transfection Reagent (cat. no. sc-29528) from Santa Cruz Biotechnology, Inc. as previously described (27).

Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Western Blot, Standard Deviation, Control

Journal: International journal of oncology

Article Title: CDC20 associated with cancer metastasis and novel mushroom‑derived CDC20 inhibitors with antimetastatic activity.

doi: 10.3892/ijo.2019.4791

Figure Lengend Snippet: Figure 3. Effect of triterpenes from P. cocos on the expression of CDC20 in chemoresistant pancreatic cancer. PANC‑1 cells were treated with PTE (30 and 60 µg/ml), or PA (30 and 60 µM), DPA (30 and 60 µM) and PPAC (30 and 60 µM), three triterpenes purified from PTE for 24 h, respectively. Whole protein extracts isolated from cells were prepared and western blot analysis with anti‑CDC20 and anti‑β‑actin antibodies was performed. β‑actin was used as a loading control. Representative images are shown. Similar results were obtained in at least two additional experiments. P. cocos, Poria cocos; CDC20, cell division cycle 20; PTE, triterpene mixture extracted from P. cocos; PA, pachymic acid; DPA, purified triterpenes dehydropachymic acid; PPAC, polyporenic acid C.

Article Snippet: The PANC-1 or MDA-MB-231 cells were transfected with human CDC20 siRNA (cat. no. sc-156154) or control siRNA-A (cat. no. sc-37007) using siRNA Transfection Reagent (cat. no. sc-29528) from Santa Cruz Biotechnology, Inc. as previously described (27).

Techniques: Expressing, Purification, Isolation, Western Blot, Control

Journal: Journal of Virology

Article Title: Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6- and E7-Expressing Keratinocytes

doi: 10.1128/jvi.00259-10

Figure Lengend Snippet: FIG. 2. Ubch10 and Cdc20 are upregulated in E6/7 cells. (A) HFKs expressing pbabe and E6/7 were treated with adriamycin for 24 h and harvested for detection of Ubch10 and Cdc20 by Western blot analysis. (B) Cells expressing E6/E7 bypass arrest in the presence of adriamycin, as indicated by positive staining for anti-BrdU. These cells also have elevated levels of Ubch10 compared to those for the controls. Magni- fication, 630.

Article Snippet: Cdc20 was immunoprecipitated from 200 g of pbabe and E6/E7 cell lysates (lysed with HLB buffer) using an agarose-conjugated mouse monoclonal antibody (Santa Cruz) and blotted with a rabbit anti-Cdc20 antibody (Santa Cruz) and monoclonal anti-BubR1 (BD Biosciences, MD).

Techniques: Expressing, Western Blot, Staining

Journal: Journal of Virology

Article Title: Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6- and E7-Expressing Keratinocytes

doi: 10.1128/jvi.00259-10

Figure Lengend Snippet: FIG. 3. Analysis of E6 and E7 alone in regulation of Cdc20, Ubch10, and cyclin B. (A) Western blot analysis of cyclin B and Ubch10 in pbabe, E6, and E6 mutant E6123-127 cells in the absence or presence of adriamycin; (B) analysis of Ubch10 and cyclin B protein levels in of cells expressing E7 or the E7 mutant E7.24 compared to those in pbabe control cells; (C) Western blot of Cdc20 levels in E6, E7, and E6/E7 cells; (D) levels of cyclin B and Ubch10 in primary human fibroblast cells expressing E6, E7, and E6/E7; (E) levels of cyclin B and Ubch10 in HFKs expressing control or shRNA targeting p53.

Article Snippet: Cdc20 was immunoprecipitated from 200 g of pbabe and E6/E7 cell lysates (lysed with HLB buffer) using an agarose-conjugated mouse monoclonal antibody (Santa Cruz) and blotted with a rabbit anti-Cdc20 antibody (Santa Cruz) and monoclonal anti-BubR1 (BD Biosciences, MD).

Techniques: Western Blot, Mutagenesis, Expressing, Control, shRNA

Journal: Journal of Virology

Article Title: Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6- and E7-Expressing Keratinocytes

doi: 10.1128/jvi.00259-10

Figure Lengend Snippet: FIG. 4. E6/E7 have an active SAC but are able to enter mitosis and degrade cyclin B (Cyc) and transverse to the next round of DNA replication. (A) E6/E7-expressing cells enter mitosis in the presence of DNA damage, as indicated by the increase in P-H3 positive cells over a period of 28 h. (B) Coimmunoprecipitation of Cdc20 and BubR1. Cdc20 was immunoprecipitated from pbabe control and E6/E7 cells and blotted for BubR1 and Cdc20. (C) FPLC size-exclusion fractions of adriamycin-treated pbabe and E6/E7 cells analyzed for levels of BubR1 and Cdc20 by Western blot analysis. (D) Western blot analysis showing BubR1 and Cdc20 levels in FPLC size-exclusion fractions of nocodazole-treated pbabe and E6/E7 cells. (E) E6/E7 cells with DNA damage are positive for BrdU and negative for cyclin B (confirming the ability of these cells to degrade cyclin B, exit mitosis, and progress through to S phase). (F) Cyclin B was immunoprecipitated and analyzed for ubiquitylation by blotting with anti-HA antibodies.

Article Snippet: Cdc20 was immunoprecipitated from 200 g of pbabe and E6/E7 cell lysates (lysed with HLB buffer) using an agarose-conjugated mouse monoclonal antibody (Santa Cruz) and blotted with a rabbit anti-Cdc20 antibody (Santa Cruz) and monoclonal anti-BubR1 (BD Biosciences, MD).

Techniques: Expressing, Immunoprecipitation, Control, Western Blot

Journal:

Article Title:

doi:

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CDC20 , Bethyl Laboratories , Cat# A301-180A, RRID:AB_890559.

Techniques: Recombinant, Software