Journal: Oncotarget

Article Title: Dietary NiCl 2 causes G 2 /M cell cycle arrest in the broiler's kidney

doi:

Figure Lengend Snippet: Antibodies used in immunohitochemistry

Article Snippet: cyclinB1 , Bioss, China , bs-0572R , 1:100.

Techniques:

Journal: Cell Cycle

Article Title: Periodic expression of Kv10.1 driven by pRb/E2F1 contributes to G2/M progression of cancer and non-transformed cells

doi: 10.1080/15384101.2016.1138187

Figure Lengend Snippet: Periodic expression of Kv10.1 along the cell cycle. A. HeLa cells were synchronized with a double thymidine block and released into fresh medium. Total cell lysates were prepared at the indicated time points and endogenous Kv10.1 was precipitated. Analysis by SDS-PAGE and Western blotting using anti-Kv10.1, anti-cyclin A2 and anti-Cyclin B1 showed that Kv10.1 expression changes along the cell cycle, with a peak expression between 8 and 12 h corresponding to G2/M. Calnexin and Actin were used as loading controls. B. Asynchronous HeLa cells were labeled with anti-Cyclin B1, anti- p-Histone H3 (Ser 28) and anti-Kv10.1. Cells in G2, as evidenced by cytoplasmic Cyclin B1 signal, as well as mitotic cells (nuclear Cyclin B1 and p-H3 Ser 28) showed Kv10.1 reactivity at the plasma membrane. C. Cyclin B1-positive cells were localized to the proliferative compartment, at the bottom and sides of the crypt. Kv10.1 positive cells were also found in the proliferative compartment of the colon crypt. White arrows indicate Kv10.1 and Cyclin B1 expressing cells. Scale bar 20 µm.

Article Snippet: The tissue sections were incubated overnight at 4°C with primary antibodies mouse anti-Kv10.1 dilution 1:100, and rabbit anti-Cyclin B1 (1:200; Acris https://www.acris-antibodies.com/cyclin-b1-ta590439.htm ).

Techniques: Expressing, Blocking Assay, SDS Page, Western Blot, Labeling

Journal: Cell Cycle

Article Title: Periodic expression of Kv10.1 driven by pRb/E2F1 contributes to G2/M progression of cancer and non-transformed cells

doi: 10.1080/15384101.2016.1138187

Figure Lengend Snippet: Kv10.1 depletion disrupts cell cycle progression in HeLa cells. A. Western blotting showed upregulated expression of Cyclins A2 and B1 upon Kv10.1 knockdown. Expression of Cyclins D1 and E1 was comparable between knockdown and control. B-D. HeLa cells transfected with siRNA control (black trace) and Kv10.1 siRNA (red trace) were synchronized with a double thymidine block and released into fresh medium. Cells were harvested at the indicated time points for FACS analysis of cell cycle profile. B. Percentage of cells in G0/G1 phase. C. Percentage of cells in S phase. D. Percentage of cells in G2/M phase. HeLa cells accumulated at G2/M upon Kv10.1 knockdown. All experiments were performed at least 3 times.

Article Snippet: The tissue sections were incubated overnight at 4°C with primary antibodies mouse anti-Kv10.1 dilution 1:100, and rabbit anti-Cyclin B1 (1:200; Acris https://www.acris-antibodies.com/cyclin-b1-ta590439.htm ).

Techniques: Western Blot, Expressing, Transfection, Blocking Assay

Journal: Cell Cycle

Article Title: Periodic expression of Kv10.1 driven by pRb/E2F1 contributes to G2/M progression of cancer and non-transformed cells

doi: 10.1080/15384101.2016.1138187

Figure Lengend Snippet: Kv10.1 depletion alters the periodicity of key G2/M regulatory proteins. HeLa cells were synchronized with a double thymidine block and released into fresh medium. Total cell lysates from siRNA control and Kv10.1 siRNA-transfected cells were prepared at the indicated time points. A. Analysis by SDS-PAGE and Western blotting of Cyclin D1 and E1 did not show differences. Levels of phosphorylated pRb rose at the same time in siRNA control and Kv10.1 siRNA transfected cells. However, pRb remained in hyperphosphorylated state for longer time in cells lacking Kv10.1. Densitograms from the blots corresponding to pRb Ser 780, pRb Ser 795 and pRb Ser 807–811 are plotted in the lower part of the panel. B. Western blotting showed that degradation of Cyclin A2 and B1 was delayed in Kv10.1 knockdown cells, as well as the dephosphorylation of pCdk1 (Y15). All experiments were performed at least 3 times.

Article Snippet: The tissue sections were incubated overnight at 4°C with primary antibodies mouse anti-Kv10.1 dilution 1:100, and rabbit anti-Cyclin B1 (1:200; Acris https://www.acris-antibodies.com/cyclin-b1-ta590439.htm ).

Techniques: Blocking Assay, Transfection, SDS Page, Western Blot, De-Phosphorylation Assay

Journal: Cell Cycle

Article Title: Periodic expression of Kv10.1 driven by pRb/E2F1 contributes to G2/M progression of cancer and non-transformed cells

doi: 10.1080/15384101.2016.1138187

Figure Lengend Snippet: Kv10.1 absence alters cell cycle progression in mouse embryonic fibroblasts. A. Western blotting showed upregulated expression of Cyclins D1, A2 and B1 in Kv10.1 knockout MEFs. Expression of Cyclin E1 was comparable between knockout and wild type. (B-D) Wild type MEFs (black trace) and knockout MEFs (red trace) were synchronized after 72 h of serum starvation and released into fresh medium. MEFs were harvested at the indicated time points for FACS analysis of cell cycle status. B. Percentage of cells in G0/G1 phase. C. Percentage of cells in S phase. D. Percentage of cells in G2/M phase. Knockout MEFs accumulated at G2/M. All experiments were performed at least 3 times.

Article Snippet: The tissue sections were incubated overnight at 4°C with primary antibodies mouse anti-Kv10.1 dilution 1:100, and rabbit anti-Cyclin B1 (1:200; Acris https://www.acris-antibodies.com/cyclin-b1-ta590439.htm ).

Techniques: Western Blot, Expressing, Knock-Out

Journal: Oncotarget

Article Title: Targeting CXCR1 on breast cancer stem cells: signaling pathways and clinical application modelling

doi:

Figure Lengend Snippet: Data are mean ± SE of three different experiments. *** p ≤ 0.0005. In (B) a representative western blotting and relative densitometric analysis, expressed as relative units, for cyclin B1 in control and treated mammospheres. Data are mean ± SE of three different experiments. ** p ≤ 0.005. In (C) immunofluorescence analysis for cyclin B1 in control and treated mammospheres, Bar = 25 μM. C: control: R: reparixin 10 μM; P: paclitaxel 5 nM; R + P: reparixin 10 μM + paclitaxel 5 nM.

Article Snippet: Antibodies anti-Cyclin D1, anti-PI3 Kinase, p27 and anti-P-FOXO3A were purchased from Abcam (Cambridge, MA, USA), anti-Cyclin B1 was from Acris Antibodies (San Diego, CA, USA), anti-NFkB and anti-P-EGFR were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and anti-β catenin was from Zymed (San Francisco, CA, USA).

Techniques: Western Blot, Immunofluorescence

Journal: Oncotarget

Article Title: Targeting CXCR1 on breast cancer stem cells: signaling pathways and clinical application modelling

doi:

Figure Lengend Snippet: In (A) cytofluorimetric analysis of cell cycle in the presence of the specific antibody administered alone or in combination with paclitaxel (P). Data are mean ± SE of three different experiments. *** p ≤ 0.0005. In (B) representative western blotting and relative densitometric analysis, expressed as relative units, for cyclin B1 and p-FAK in the same experimental conditions. Data are mean ± SE of three different experiments. * p ≤ 0.05; ** p ≤ 0.005; *** p ≤ 0.0005.

Article Snippet: Antibodies anti-Cyclin D1, anti-PI3 Kinase, p27 and anti-P-FOXO3A were purchased from Abcam (Cambridge, MA, USA), anti-Cyclin B1 was from Acris Antibodies (San Diego, CA, USA), anti-NFkB and anti-P-EGFR were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and anti-β catenin was from Zymed (San Francisco, CA, USA).

Techniques: Western Blot

Journal: American Journal of Cancer Research

Article Title: FOXA1 transcriptionally up-regulates cyclin B1 expression to enhance chondrosarcoma progression

doi:

Figure Lengend Snippet: Knockdwon of cyclin B1 inhibits tumorigenicity of chondrosarcoma. A. CH2879 cells were infected with lentivirus expressing control or FOXA1 shRNA. qRT-PCR of CCNB1 gene expression in CH2879 with control or FOXA1 shRNA. B. CH2879 cell lines stably expressing vector control or FOXA1 were used in this assay. qRT-PCR of CCNB1 gene expression in CH2879 stable cell lines. C and D. Lysates of CH2879 cells were immunoblotted as indicated antibodies. Actin served as the control. E. MTT assay was performed to assess cell proliferation at different time points including 24, 48, 72 and 96 hr. F. Images and quantification of colony forming ability assay of CH2879 cells. G. CH2879 cell lines stably expressing vector control or FOXA1 were infected with control or two specific cyclin B1 shRNA. Lysates were harvested and immunoblotted. H. MTT assay of indicated cells was determined for cell proliferation at different time points including 24, 48, 72, and 96 hr. Data are means ± SD, Student’s t-test. *P < 0.05, **P < 0.01. ***P < 0.001.

Article Snippet: Whole-cell lysates were analyzed with the following antibodies: FOXA1 (GeneTex, GTX100308), p53 (Cell Signaling, #2524), p21 (CALBIOCHEM, OP64), CCNB1 (GeneTex, GTX100911), and β-actin (GeneTex, GTX109639).

Techniques: Infection, Expressing, Control, shRNA, Quantitative RT-PCR, Gene Expression, Stable Transfection, Plasmid Preparation, MTT Assay

Journal: American Journal of Cancer Research

Article Title: FOXA1 transcriptionally up-regulates cyclin B1 expression to enhance chondrosarcoma progression

doi:

Figure Lengend Snippet: FOXA1 binds specifically to the CCNB1 promoter in chondrosarcoma cells. A. The FOXA1 binding motif on CCNB1 promoter was predicted by PROMO 3.0 and is visually represented in WebLogo (http://weblogo.berkeley.edu). A schematic of the FOXA1 binding site located in CCNB1 promoter region within -1000 and -400 bp to transcription starting site. B. Luciferase reporter assay of the promoter activities of CCNB1 promoter-deletion mutants with or without ectopic expression of FOXA1 in 293 cells. C. One FOXA1 binding site located in the CCNB1 promoter within -400 bp to transcription starting site was mutated by site direct mutagenesis. Reporter assay of CCNB1 promoter activity with FOXA1-binding site mutations in 293 cells ectopically expressing vector control or FOXA1. D. CH2879 cells stably expressing vector control or FOXA1 were subjected to ChIP assay. Cross-linked chromatin was immunoprecipitated by utilizing FOXA1 or IgG antibodies. The input and immunoprecipitated DNA were subjected to q-PCR primers corresponding to the promoter region of CCNB1 (Table S1). P values were determined by Student’s t-test, error bars represent ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Whole-cell lysates were analyzed with the following antibodies: FOXA1 (GeneTex, GTX100308), p53 (Cell Signaling, #2524), p21 (CALBIOCHEM, OP64), CCNB1 (GeneTex, GTX100911), and β-actin (GeneTex, GTX109639).

Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Mutagenesis, Activity Assay, Plasmid Preparation, Control, Stable Transfection, Immunoprecipitation

Journal: American Journal of Cancer Research

Article Title: FOXA1 transcriptionally up-regulates cyclin B1 expression to enhance chondrosarcoma progression

doi:

Figure Lengend Snippet: Knockdown of FOXA1 attenuates chondrosarcoma tumor growth in vivo. A. Representative images of tumors harvested from subcutaneous mice xenografted with CH2879-control or FOXA1-knockdown cells. B. Tumor volume was monitored every 3 days and plotted. n = 4 per group. C. A proposed model of FOXA1 transcriptionally mediated chondrosarcoma cell cycle progress. In chondrosarcoma cells, FOXA1 binds specifically to the indicated region of CCNB1 promoter to facilitate its expression and thereby promote G2/M transition via cycllinB1-CDK1 complex activation in the cell cycle. Data are shown as means ± SD; Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Whole-cell lysates were analyzed with the following antibodies: FOXA1 (GeneTex, GTX100308), p53 (Cell Signaling, #2524), p21 (CALBIOCHEM, OP64), CCNB1 (GeneTex, GTX100911), and β-actin (GeneTex, GTX109639).

Techniques: Knockdown, In Vivo, Control, Expressing, Activation Assay