Journal: British Journal of Cancer

Article Title: Gene expression profile and response to trastuzumab–docetaxel-based treatment in breast carcinoma

doi: 10.1038/sj.bjc.6605310

Figure Lengend Snippet: References and nucleotide sequences of primers and probes used in this study

Article Snippet: Cell cycle , cdc27 , NM_001256 , Hs01559427_m1.

Techniques:

Journal: Genes & Cancer

Article Title: Chronic low dose arsenic exposure preferentially perturbs mitotic phase of the cell cycle

doi: 10.18632/genesandcancer.185

Figure Lengend Snippet: A. The phosphorylated BubR1 and Cdc27 in the cells, after releasing from nocodazole block, were analyzed by immunoblotting analysis. B. After blocking protein synthesis by CHX, the levels of cln B1 expression in the cells were tested at different time points of nocodazole releasing. C. After adding actinomycin D (ATC) to block DNA synthesis, the cells were synchronized in mitosis by nocodazole treatment. Following releasing from mitosis block, quantitative PCR was performed to determine the levels of cln B1 expression. Error bars represent SD from 5 experiments ( p < 0.05).

Article Snippet: Cdc27 antibody was from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Blocking Assay, Western Blot, Expressing, DNA Synthesis, Real-time Polymerase Chain Reaction

Journal: Genes & Cancer

Article Title: Chronic low dose arsenic exposure preferentially perturbs mitotic phase of the cell cycle

doi: 10.18632/genesandcancer.185

Figure Lengend Snippet: A. After being treated with sc or siRNA-Plk1 , the suppression of Plk1 expression in the cells was tested by immunoblotting (left panels). After the treatment of scRNA- or siRNA-Plk1 , the expressions of the phosphorylated BubR1 and Cdc27 were examined in the cells that were released 1 h from nocodazole block (right panels). B. Six hours after released from nocodazole block, the percentages of the cells in mitotic phase were measured by flow cytometry. Error bars are the SD over 5 experiments ( n = 5; p < 0.01).

Article Snippet: Cdc27 antibody was from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Expressing, Western Blot, Blocking Assay, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Spindle Assembly Checkpoint Protein Cdc20 Transcriptionally Activates Expression of Ubiquitin Carrier Protein UbcH10

doi: 10.1074/jbc.m110.160671

Figure Lengend Snippet: FIGURE 4. Cdc20, APC/C, and CBP/p300 occupy UBCH10 promoter and cause chromatin remodeling. A, Cdc20 occupies the UBCH10 promoter in UPCI:SCC104 cells. ChIP assay was done using antibodies specific for RNA pol II and Cdc20 and no antibody as control. Precipitated DNA was PCR- amplified using primers encompassing 322- to 39-nt region of UBCH10 promoter relative to the transcription start site. B, Cdc20 and CBP occupy the UBCH10 promoter in HepG2 cells. ChIP assay was performed using antibodies specific for RNA pol II, Cdc20, and CBP and normal IgG as control. Precipitated DNA was PCR-amplified as indicated in A. C, APC/C is present on UBCH10 promoter in HepG2 cells. ChIP assay was performed using antibodies specific for RNA pol II, Cdc20, CBP, p300, Cdc27, and normal IgG as control. Precipitated DNA was PCR-amplified as indicated in A. D, MCC is not present on UBCH10 promoter. ChIP assay was performed using antibodies specific for RNA pol II, Cdc20, CBP, Mad2, and normal IgG as control. Precipitated DNA was PCR-amplified as indicated in A. E, ectopic expression of Cdc20 enhances histone acetylation of the UBCH10 promoter. HepG2 cells were transiently transfected with pCDNA5-FLAG-CDC20 or empty vector as control. Chromatin immunoprecipitation was done using antibodies specific for RNA pol II, Cdc20-acetylated histone 3 (Lys-9/14), and normal IgG as control. Precipitated chromatin was estimated by quantitative real time PCR. The results are expressed as percent of input. Bars represent mean S.E. of two independent determinations from two separate chromatin preparations. F, knockdown of Cdc20 reduces histone acetylation of UBCH10 promoter. HepG2 cells were transiently transfected with siRNA oligos to Cdc20 mRNA or scrambled siRNA oligo as control. Precipitated DNA was PCR-amplified as indicated in A.

Article Snippet: Various siRNA constructs directed against Cdc20 (sc-36160, Santa Cruz Biotechnology, used in Figs. 1, C andD, 2,D and E, 4F, and 5,A andC, and supplemental Fig. S6; catalog no. AM 16706, Ambion (Austin, TX) used in Figs. 1, E and G, 2G, and Fig. 6, B and C, and supplemental Fig. S7), UbcH10 (Santa Cruz Biotechnology), Cdc27 and Mad2 (Santa Cruz Biotechnology), and scrambled control (Ambion) were used at a final concentration of 80 nM.

Techniques: Control, Amplification, Expressing, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Knockdown

Journal: Journal of Biological Chemistry

Article Title: Spindle Assembly Checkpoint Protein Cdc20 Transcriptionally Activates Expression of Ubiquitin Carrier Protein UbcH10

doi: 10.1074/jbc.m110.160671

Figure Lengend Snippet: FIGURE 5. Cdc20 regulates APC/C-CBP/p300 interaction and influences their recruitment to the UBCH10 promoter. A, APC/C-CBP interaction is reduced in HepG2 cells upon Cdc20 knockdown. HepG2 cells were transiently transfected with siRNA oligos to Cdc20 mRNA or scrambled siRNA oligo as control. Whole cell extracts were immunoprecipitated (IP) with antibodies specific for Cdc27 or CBP or normal IgG. Immunocomplexes and input (20% of the whole cell extracts) were probed with antibodies to the indicated proteins. WB, Western blot. B, APC/C and CBP recruitment to the UBCH10 promoter is reduced upon Cdc20 knockdown. HepG2 cells were transiently transfected with siRNA oligos to Cdc20 mRNA or scrambled siRNA as control. ChIP assay was done using antibodies specific for Cdc20, Cdc27, and CBP and normal IgG as control. Precipitated DNA was PCR-amplified using primers encompassing 322- to 39-nt region of the UBCH10 promoter relative to the transcription start site. C, Cdc20 and CBP recruitment to the UBCH10 promoter is reduced upon Cdc27 knockdown. HepG2 cells were transiently transfected with siRNA oligos to Cdc27 mRNA or scrambled control. ChIP assay was performed using antibodies specific for Cdc20, Cdc27, and CBP and IgG control. Precipitated DNA was PCR-amplified using primers as indicated in B. D, knockdown of Cdc27 suppresses UBCH10 promoter activity. HepG2 cells were transiently co-transfected with pSN1 (100 ng) and siRNA oligos directed against Cdc27 mRNA or scrambled siRNA as control. Protein lysates were prepared for luciferase assay. The data represent three independent determinations (average S.E.). Lysates were also subjected to Western blot analysis with antibodies against Cdc27 and -actin. E, ectopic expression of Cdc20 does not activate UBCH10 promoter in Cdc27 knockdowncells.HepG2cellsweretransientlyco-transfectedwithpSN1(100ng)andpCDNA5-FLAG-CDC20and/orsiRNAoligosdirectedagainstCdc27mRNA or scrambled control. Protein lysates were isolated for luciferase assay. The data represent three independent determinations (average S.E.). Lysates were also subjected to Western blot analysis with antibodies against FLAG, Cdc27, and -actin.

Article Snippet: Various siRNA constructs directed against Cdc20 (sc-36160, Santa Cruz Biotechnology, used in Figs. 1, C andD, 2,D and E, 4F, and 5,A andC, and supplemental Fig. S6; catalog no. AM 16706, Ambion (Austin, TX) used in Figs. 1, E and G, 2G, and Fig. 6, B and C, and supplemental Fig. S7), UbcH10 (Santa Cruz Biotechnology), Cdc27 and Mad2 (Santa Cruz Biotechnology), and scrambled control (Ambion) were used at a final concentration of 80 nM.

Techniques: Knockdown, Transfection, Control, Immunoprecipitation, Western Blot, Amplification, Activity Assay, Luciferase, Expressing, Isolation