Fig 1: The ubiquitination of p65 by APC11CDC20 AImmunoblot of Flag‐p65‐linked ubiquitination promoted by Myc‐CDC20. HEK293T cells were co‐transfected with Flag‐p65, HA‐Ubiquitin, with or without Myc‐CDC20 or vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.BImmunoblot of Flag‐p65‐linked ubiquitination promoted by HA‐APC11. HEK293T cells were transfected with Flag‐p65, His‐Ubiquitin, with or without HA‐APC11 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.C, DImmunoblot of Flag‐p65‐linked in vitro ubiquitination promoted by GST‐CDC20 (C) or GST‐APC11 (D). Bacterially expressed and purified GST‐CDC20 or GST‐APC11 were incubated with purified proteins, including HA‐Ubiquitin, E1, E2, and Flag‐p65 as indicated at 32°C for 1 h.EImmunoblot of Flag‐p65‐linked ubiquitination promoted by truncated Myc‐CDC20 fragments. HEK293T cells were transfected with Flag‐p65, HA‐Ubiquitin, with or without Myc‐CDC20 171‐499 fragment (containing WD40 domain), Myc‐CDC20 1–170 fragment (WD40 domain deficient), and vector plasmids. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.FImmunoblot of Flag‐p65‐linked ubiquitination promoted by HA‐APC11 in NC and CDC20sh cells. NC or CDC20sh HEK293T cells were co‐transfected with Flag‐p65, His‐Ubiquitin, with or without HA‐APC11 and vector plasmids. Transfected cells were treated with 10 μM MG132 for 6 h before collection.
Fig 2: CDC20 regulated osteogenic differentiation of BMSCs in a p65‐dependent manner, related to Fig 8 AFluorescent staining of lentiviruses injected from tail intravenously.B, CThe efficiency of p65 knockdown determined by qRT–PCR (B) and Western blot (C) of BMSCs in Sp7‐Cre;Cdc20f / f mice (n = 6).D, EThe ALP staining (D) and ALP quantification (E) of control and CDC20 knockdown hBMSCs after 7 days osteogenic differentiation treated with negative control or p65si RNA (n = 5).F, GThe ALP staining (F) and ALP quantification (G) of NC and CDC20sh hBMSCs after 7 days osteogenic differentiation treated with BAY 11–7,082 (n = 6).HThe expression of RUNX2 in BMSCs from Cdc20f / f mice and Sp7‐Cre;Cdc20f / f mice after 7 days osteogenic differentiation treated with negative control or p65si, determined by qRT–PCR (n = 5). Data information: Data are displayed as mean ± SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one‐way ANOVA followed by independent two‐tailed Student’s t‐tests or a Tukey’s post hoc test and defined as *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: Knockdown of CDC20 enhances adipogenic differentiation of hBMSCs. A Fluorescence micrographs showing the lentivirus transduction efficiency. Scale bar, 500 μm. B CDC20 mRNA expression in control shRNA (NC) and CDC20 knockdown (shCDC20-1, shCDC20-2) groups examined using qRT-PCR. (C, D) Oil red O staining C and quantification D of hBMSCs after adipogenic induction for 21 days. Scale bar, 100 μm. (E, F) Adipogenic regulators PPARG E and CEBPA F mRNA expression levels determined by qRT-PCR after adipogenic induction for 14 days. (G, H) Western blotting analysis G and quantification H of CDC20, PPARγ, C/EBPα, and the internal control GAPDH of negative control (NC) and CDC20 knockdown (shCDC20) cells in proliferation medium (PM) and adipogenic medium (AM) for 7 days. All data are presented as the mean ± SD (n = 3, **P < 0.01, ***P < 0.001)
Fig 4: Mechanisms of CCT4 regulating the growth of HCC. (A) Cdc20 could be determined in IP lysate of CCT4 protein, and CCT4 could be detected in IP lysate of Cdc20 protein. (B) Securin and cyclin D1 were determined by WB in Huh7 cells containing shRNA of CCT4 and control. (C) Statistical analysis of securin and cyclin D1 expression. (D) Bim, Bcl-2, caspase 9, and cleaved caspase 9 were detected by WB in Huh7 cells containing shRNA of CCT4 and control. (E) Statistical analysis of Bim, Bcl-2, caspase 9, and cleaved caspase 9 expression. (F) Model of CCT4 influencing the cell proliferation and apoptosis. ∗P < 0.01, †P < 0.05. APC: Anaphase-promoting complex; Bcl-2: B-cell lymphoma-2; Bim: Bcl-2 interacting mediator of cell death; CCT4: Chaperonin containing t-complex 4; HCC: Hepatocellular carcinoma; IgG: Immunoglobulin G; IP: Immunoprecipitation; MCC: Mitotic checkpoint complex; shRNA: Short-hairpin RNA; WB: Western blot.
Fig 5: CDC20, CNNB2, and BUB1 affects the proliferation of SK-NEP-1 cells. (A-D) The cell proliferative ability was assessed by the Cell Counting Kit-8 (CCK-8), and EdU assay after CDC20, CNNB2, and BUB1 was knocked down in SK-NEP-1 cells. (E-H) Down-regulation of CDC20, CNNB2, and BUB1 arrests the cell cycle of SK-NEP-1 cells(**P < 0.01, ns, none sense).
Supplier Page from Abcam for Anti-Cdc20 antibody