Journal: Nature cell biology

Article Title: Evidence for how APC-C-Cdc20 changes its substrate specificity in mitosis

doi: 10.1038/ncb2165

Figure Lengend Snippet: APC10 is required to recruit SAC-sensitive substrates ( a ) Depletion of APC10. Cells were treated with siRNA oligos against GAPDH (control) or APC10 and the results from 2 independent experiments analyzed as in . Hsp70 was used as a loading control. ( b and c ) APC10 is required for Cyclin B1 but not for Cyclin A degradation. Cells were treated with siRNA oligos against APC10, injected with a plasmid encoding Cyclin A-Venus ( b ) or Cyclin B1-Venus ( c ) and analysed by time-lapse as . The mean +-− s.d. for all cells is plotted in black (siAPC10) and the values from the control cells are plotted in grey (siCTR). n = number of cells analyzed from 2 independent experiments. ( d and e ) APC10 mediates Cyclin B1 and Cdc20 but not Cyclin A binding to APC-C. HeLa cells were treated with siRNA against GAPDH (CTR) or APC10 and synchronised in metaphase as in . The APC-C complex was immunoprecipitated with anti-APC4 antibodies and samples blotted for APC3, APC4, APC10, Cdc20, Cyclin A and Cyclin B1 ( d ). ( e ) Bar diagrams show the remaining amount of APC10 and the amount of Cyclin B1 and Cdc20 bound to the metaphase APC-C quantified using a LI-COR Odyssey scanner and normalised to the level of APC4. Levels of the proteins bound to control APC-C were set to 1. The mean +/− s.d. of 3 experiments is shown.

Article Snippet: Cdc20 (A301-180A, Bethyl laboratories) 1:200, BubR1 (A300-386A, Bethyl laboratories), 1:500, Cyclin B1 (mAb GNS-1, BD Pharmingen) 1:1000, Cyclin A (mAb AT10.3, CRUK) 1:1000, APC3 (610455, BD Transduction Laboratories) 1:500, APC4 (monoclonal antibody raised against a C-terminal peptide) 1:500, APC11 (monoclonal antibody raised against a C-terminal peptide) 1:500, APC7 (Abcam 4171) 1:500, APC8 (Biolegend) 1:500, APC6 (Santa Cruz Biotechnology), APC10 (raised against full length protein) 1:1000, Hsp70 (Sigma) 1:5000.

Techniques: Control, Injection, Plasmid Preparation, Binding Assay, Immunoprecipitation

Journal: Nature cell biology

Article Title: Evidence for how APC-C-Cdc20 changes its substrate specificity in mitosis

doi: 10.1038/ncb2165

Figure Lengend Snippet: APC3 is only required to bind Cdc20 when the SAC is satisfied ( a - d ) APC3 is only required to bind Cdc20 when the SAC is satisfied. HeLa cells were treated with siRNA against GAPDH (CTR) or APC3 and synchronised in prometaphase by treating with 100 ng-ml nocodazole plus 10 μM MG132 to stabilise Cyclin A ( a & b ), or synchronised in prometaphase with 100 ng-ml nocodazole, released into medium containing 10 μM MG132 and incubated for a further 3 hrs to obtain metaphase cells ( c & d ). The APC-C complex was immunoprecipitated with anti-APC4 antibodies and samples blotted for APC3, APC4, Cdc20, BubR1 and Cyclin A ( a ) or Cyclin B1 ( b ). ( b and d ) Bar diagrams show the remaining amount of APC3 and the amount of Cdc20 and Cyclin A or B1 bound to the prometaphase APC-C ( b ) or the metaphase APC-C ( d ), quantified using a LI-COR Odyssey scanner and normalised to the level of APC4. Levels of the proteins bound to control APC-C were set to 1. Error bars shown are mean+/− s.d. of 3 experiments. ( e ) APC3 is required for free Cdc20 to bind to the APC-C. Cells were treated with siRNA oligos against GAPDH or APC3 and synchronized as in ( a ). Purified recombinant His 6 -tagged Cdc20 was added to the cell extracts and the APC-C was immunoprecipitated with an anti-APC4 antibody. Samples were blotted for APC3, APC4 and Cdc20. Recombinant Cdc20 runs at a higher molecular mass than endogenous Cdc20.

Article Snippet: Cdc20 (A301-180A, Bethyl laboratories) 1:200, BubR1 (A300-386A, Bethyl laboratories), 1:500, Cyclin B1 (mAb GNS-1, BD Pharmingen) 1:1000, Cyclin A (mAb AT10.3, CRUK) 1:1000, APC3 (610455, BD Transduction Laboratories) 1:500, APC4 (monoclonal antibody raised against a C-terminal peptide) 1:500, APC11 (monoclonal antibody raised against a C-terminal peptide) 1:500, APC7 (Abcam 4171) 1:500, APC8 (Biolegend) 1:500, APC6 (Santa Cruz Biotechnology), APC10 (raised against full length protein) 1:1000, Hsp70 (Sigma) 1:5000.

Techniques: Incubation, Immunoprecipitation, Control, Purification, Recombinant

Journal: Nature cell biology

Article Title: Evidence for how APC-C-Cdc20 changes its substrate specificity in mitosis

doi: 10.1038/ncb2165

Figure Lengend Snippet: A point mutation in APC8 is sufficient to reduce the binding of Cdc20 in prometaphase ( a ) Schematic structure of human APC8. TPR domains are shown as grey boxes. ( b ) The N338A mutation reduces Cdc20 binding to prometaphase APC-C. HeLa cells with an inducible wild type Flag-APC8 (WT) or mutant Flag-APC8 N338A were treated with siRNA against GAPDH (CTR) or APC8 and synchronised in prometaphase as in . The APC-C was immunoprecipitated using anti-APC4 antibodies and the immunoprecipitates blotted with antibodies against APC2, APC3, APC4, APC6, APC7, APC8, APC10, APC11 and Cdc20 and the extent of depletion measured by quantitative immunoblotting. The asterisk indicates endogenous APC8. ( c ) Quantification of APC3, Cdc20 and Flag-APC8 bound to the APC-C by quantitative immunoblotting and normalised to the level of APC4. Levels of the proteins bound to control APC-C were set to 1. The mean +/− s.d. of 4 independent experiments is shown. ( d ) Analysis of APC-C by size-exclusion chromatography. Control (bottom) and experimental HeLa cells expressing an inducible wild type Flag-APC8 (WT, top) or mutant Flag-APC8 N338A (middle) were treated with siRNA oligos against APC8 and synchronised in prometaphase as in panel b and extracts prepared as in . Cell extracts were fractioned on a Superose 6 column and fractions were blotted with antibodies against APC3, APC4, Flag epitope, APC8, Cdc20 and BubR1. The peak of APC-C migration is indicated by the black bar.

Article Snippet: Cdc20 (A301-180A, Bethyl laboratories) 1:200, BubR1 (A300-386A, Bethyl laboratories), 1:500, Cyclin B1 (mAb GNS-1, BD Pharmingen) 1:1000, Cyclin A (mAb AT10.3, CRUK) 1:1000, APC3 (610455, BD Transduction Laboratories) 1:500, APC4 (monoclonal antibody raised against a C-terminal peptide) 1:500, APC11 (monoclonal antibody raised against a C-terminal peptide) 1:500, APC7 (Abcam 4171) 1:500, APC8 (Biolegend) 1:500, APC6 (Santa Cruz Biotechnology), APC10 (raised against full length protein) 1:1000, Hsp70 (Sigma) 1:5000.

Techniques: Mutagenesis, Binding Assay, Immunoprecipitation, Western Blot, Control, Size-exclusion Chromatography, Expressing, FLAG-tag, Migration

Journal: Nature cell biology

Article Title: Evidence for how APC-C-Cdc20 changes its substrate specificity in mitosis

doi: 10.1038/ncb2165

Figure Lengend Snippet: The Cdc20-binding site on APC8 is also required in metaphase ( a and b ) Cyclin B1 degradation is inhibited in cells expressing APC8 N338A . siRNA against APC8 and a plasmid encoding Cyclin B1-Venus were transfected into the HeLa cells with an inducible siRNA-resistant wild type Flag-APC8 ( a ) or mutant Flag-APC8 N338A ( b ) and analysed as in . Mean +/− s.d. values are shown. n = number of cells analyzed from 3 independent experiments. ( c ) APC8 is important for free-Cdc20 to bind to the APC-C. HeLa cells expressing an inducible wild type Flag-APC8 (WT) or mutant Flag-APC8 N338A were treated with siRNA against GAPDH (CTR) or APC8 as in and synchronised in metaphase as in . The APC-C was immunoprecipitated using anti-APC4 antibodies and the immunoprecipitates blotted with antibodies against APC4, APC8 and Cdc20. The asterisk indicates endogenous APC8. ( d ) APC8 N338A mutation reduces APC-C activity in vitro . The APC-C incorporating Flag-APC8 or Flag-APC8 N338A was prepared as in and its activity assayed using securin as a substrate in an in vitro ubiquitination reaction as previously described . ( e ) Model for APC-C regulation during early mitosis. In prometaphase (left), the APC-C recognizes substrates such as Cyclin A through the APC3 subunit but interacts through APC8 with Cdc20, as co-activator or as part of the SAC complex. The SAC proteins associated with Cdc20 could prevent Cdc20 accessing its APC3 binding site. In metaphase (right), the APC-C recognizes substrates such as Cyclin B1 through APC10 and Cdc20 forming a bi-partite receptor, and Cdc20 requires both APC3 and APC8 to interact with and activate the APC-C.

Article Snippet: Cdc20 (A301-180A, Bethyl laboratories) 1:200, BubR1 (A300-386A, Bethyl laboratories), 1:500, Cyclin B1 (mAb GNS-1, BD Pharmingen) 1:1000, Cyclin A (mAb AT10.3, CRUK) 1:1000, APC3 (610455, BD Transduction Laboratories) 1:500, APC4 (monoclonal antibody raised against a C-terminal peptide) 1:500, APC11 (monoclonal antibody raised against a C-terminal peptide) 1:500, APC7 (Abcam 4171) 1:500, APC8 (Biolegend) 1:500, APC6 (Santa Cruz Biotechnology), APC10 (raised against full length protein) 1:1000, Hsp70 (Sigma) 1:5000.

Techniques: Binding Assay, Expressing, Plasmid Preparation, Transfection, Mutagenesis, Immunoprecipitation, Activity Assay, In Vitro, Ubiquitin Proteomics

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