Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Western blot analysis. HeLa and SiHa cells with or without pre-incubation (1 h) with 5 µM NAC were treated by icaritin for 24 h. Multiple SDS-PAGE gels were prepared for each sample, and each gel was used for the detection of one protein. Thus, one loading control, β-actin, was used for all proteins of each sample. (A) Detection of MMP-9. (B) Detection of cell cycle-associated proteins. (C) Detection of apoptosis-associated proteins. (D) Quantification of apoptosis-associated proteins in the 0 µM, 2× IC 50 and 2× IC 50 + NAC groups. Relative protein levels were quantified from western blots using ImageJ and presented as ratios of each protein band relative to the loading control. (*P<0.05, **P<0.01, ***P<0.001 vs. 0 µM). NAC, N-acetyl cysteine, MMP-9, matrix metalloproteinase-9; CDK1, cyclin-dependent kinase 1; CDC25C, cell division cycle 25C; XIAP, X-linked inhibitor of apoptosis protein; Bax, apoptosis regulator Bax; Bcl-2, B-cell lymphoma-2.

Article Snippet: The membranes were blocked in Tris-buffered saline with Tween-20 [10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween-20; PBST) containing 5% (w/v) nonfat milk for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C [B-cell lymphoma 2 (Bcl-2; 1:500; cat. no. sc-7382) and apoptosis regulator Bax (Bax; 1:500; cat. no. sc-7480) from Santa Cruz Biotechnology, Inc.; cleaved caspase 3 (1:500; cat. no. ab2302), cleaved caspase 9 (1:200; cat. no. ab2324) and X-linked inhibitor of apoptosis protein (XIAP; 1:1,000; cat. no. ab28151) from Abcam (Cambridge, UK); cyclin-dependent kinase 1 (CDK1)-pT14 (1:1,000; cat. no. bs-3091R), cell division cycle 25C (CDC25C)-pS216 (1:1,000; cat. no. bs-3096R), β-actin (1:5,000; cat. no. bs-0061R) and matrix metalloproteinase 9 (MMP9; 1:1,000; cat. no. bs-4593R) from BIOSS].

Techniques: Western Blot, Incubation, SDS Page

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A Robust Protocol to Map Binding Sites of the 14-3-3 Interactome: Cdc25C Requires Phosphorylation of Both S216 and S263 to bind 14-3-3 *

doi: 10.1074/mcp.M110.005157

Figure Lengend Snippet: Identification of new sites required for 14-3-3 binding to Cdc25C. A, Top panel: schematic diagram of the domains and motifs found in human Cdc25C including the known site Ser216. Additional potential sites were scored using matrices S/T-L/M and S/T-x-P (supplementary Fig. S4), or Scansite 14-3-3. Top scoring results are listed and those tested by in situ binding are indicated. Sites marked by * are the Cdc25B homologs to the Cdc25C sequence directly above. Mammalian Cdc25C is widely reported as a 14-3-3 binding protein. Middle panel: Alignment of the identified 14-3-3 binding sites and overlapping NLS regions in human and Xenopus Cdc25C, compared with the same region in human Cdc25B. Identical residues are indicated in bold, similar residues in bold gray and known phosphorylated residues in red. The reported nuclear localization sequence (40) and start of phosphatase domain are indicated. Bottom panel: full-length wild-type Cdc25C or the S247A and S263A mutants were immunoprecipitated from unsynchronized cells and tested for endogenous 14-3-3 proteins. Western blots were performed on control or calyculin A treated cells. These results are representative of three independent experiments. B, Modification of Cdc25C pS216 site is unaffected by mutation of S247A and S263A. Cell lysates as in (A) were probed by anti-pS216 Cdc25C antibody (n = 3).

Article Snippet: Levels of cell division cycle protein 25C (Cdc25C) 1 pS216 were assessed using anti-pS216 monoclonal antibody (Santa Cruz Biotechnology).

Techniques: Binding Assay, In Situ, Sequencing, Immunoprecipitation, Western Blot, Modification, Mutagenesis

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Western blot analysis. HeLa and SiHa cells with or without pre-incubation (1 h) with 5 µM NAC were treated by icaritin for 24 h. Multiple SDS-PAGE gels were prepared for each sample, and each gel was used for the detection of one protein. Thus, one loading control, β-actin, was used for all proteins of each sample. (A) Detection of MMP-9. (B) Detection of cell cycle-associated proteins. (C) Detection of apoptosis-associated proteins. (D) Quantification of apoptosis-associated proteins in the 0 µM, 2× IC 50 and 2× IC 50 + NAC groups. Relative protein levels were quantified from western blots using ImageJ and presented as ratios of each protein band relative to the loading control. (*P<0.05, **P<0.01, ***P<0.001 vs. 0 µM). NAC, N-acetyl cysteine, MMP-9, matrix metalloproteinase-9; CDK1, cyclin-dependent kinase 1; CDC25C, cell division cycle 25C; XIAP, X-linked inhibitor of apoptosis protein; Bax, apoptosis regulator Bax; Bcl-2, B-cell lymphoma-2.

Article Snippet: The membranes were blocked in Tris-buffered saline with Tween-20 [10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween-20; PBST) containing 5% (w/v) nonfat milk for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C [B-cell lymphoma 2 (Bcl-2; 1:500; cat. no. sc-7382) and apoptosis regulator Bax (Bax; 1:500; cat. no. sc-7480) from Santa Cruz Biotechnology, Inc.; cleaved caspase 3 (1:500; cat. no. ab2302), cleaved caspase 9 (1:200; cat. no. ab2324) and X-linked inhibitor of apoptosis protein (XIAP; 1:1,000; cat. no. ab28151) from Abcam (Cambridge, UK); cyclin-dependent kinase 1 (CDK1)-pT14 (1:1,000; cat. no. bs-3091R), cell division cycle 25C (CDC25C)-pS216 (1:1,000; cat. no. bs-3096R), β-actin (1:5,000; cat. no. bs-0061R) and matrix metalloproteinase 9 (MMP9; 1:1,000; cat. no. bs-4593R) from BIOSS].

Techniques: Western Blot, Incubation, SDS Page

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Western blot analysis. HeLa and SiHa cells with or without pre-incubation (1 h) with 5 µM NAC were treated by icaritin for 24 h. Multiple SDS-PAGE gels were prepared for each sample, and each gel was used for the detection of one protein. Thus, one loading control, β-actin, was used for all proteins of each sample. (A) Detection of MMP-9. (B) Detection of cell cycle-associated proteins. (C) Detection of apoptosis-associated proteins. (D) Quantification of apoptosis-associated proteins in the 0 µM, 2× IC 50 and 2× IC 50 + NAC groups. Relative protein levels were quantified from western blots using ImageJ and presented as ratios of each protein band relative to the loading control. (*P<0.05, **P<0.01, ***P<0.001 vs. 0 µM). NAC, N-acetyl cysteine, MMP-9, matrix metalloproteinase-9; CDK1, cyclin-dependent kinase 1; CDC25C, cell division cycle 25C; XIAP, X-linked inhibitor of apoptosis protein; Bax, apoptosis regulator Bax; Bcl-2, B-cell lymphoma-2.

Article Snippet: The membranes were blocked in Tris-buffered saline with Tween-20 [10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween-20; PBST) containing 5% (w/v) nonfat milk for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C [B-cell lymphoma 2 (Bcl-2; 1:500; cat. no. sc-7382) and apoptosis regulator Bax (Bax; 1:500; cat. no. sc-7480) from Santa Cruz Biotechnology, Inc.; cleaved caspase 3 (1:500; cat. no. ab2302), cleaved caspase 9 (1:200; cat. no. ab2324) and X-linked inhibitor of apoptosis protein (XIAP; 1:1,000; cat. no. ab28151) from Abcam (Cambridge, UK); cyclin-dependent kinase 1 (CDK1)-pT14 (1:1,000; cat. no. bs-3091R), cell division cycle 25C (CDC25C)-pS216 (1:1,000; cat. no. bs-3096R), β-actin (1:5,000; cat. no. bs-0061R) and matrix metalloproteinase 9 (MMP9; 1:1,000; cat. no. bs-4593R) from BIOSS].

Techniques: Western Blot, Incubation, SDS Page

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Western blot analysis. HeLa and SiHa cells with or without pre-incubation (1 h) with 5 µM NAC were treated by icaritin for 24 h. Multiple SDS-PAGE gels were prepared for each sample, and each gel was used for the detection of one protein. Thus, one loading control, β-actin, was used for all proteins of each sample. (A) Detection of MMP-9. (B) Detection of cell cycle-associated proteins. (C) Detection of apoptosis-associated proteins. (D) Quantification of apoptosis-associated proteins in the 0 µM, 2× IC 50 and 2× IC 50 + NAC groups. Relative protein levels were quantified from western blots using ImageJ and presented as ratios of each protein band relative to the loading control. (*P<0.05, **P<0.01, ***P<0.001 vs. 0 µM). NAC, N-acetyl cysteine, MMP-9, matrix metalloproteinase-9; CDK1, cyclin-dependent kinase 1; CDC25C, cell division cycle 25C; XIAP, X-linked inhibitor of apoptosis protein; Bax, apoptosis regulator Bax; Bcl-2, B-cell lymphoma-2.

Article Snippet: The membranes were blocked in Tris-buffered saline with Tween-20 [10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween-20; PBST) containing 5% (w/v) nonfat milk for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C [B-cell lymphoma 2 (Bcl-2; 1:500; cat. no. sc-7382) and apoptosis regulator Bax (Bax; 1:500; cat. no. sc-7480) from Santa Cruz Biotechnology, Inc.; cleaved caspase 3 (1:500; cat. no. ab2302), cleaved caspase 9 (1:200; cat. no. ab2324) and X-linked inhibitor of apoptosis protein (XIAP; 1:1,000; cat. no. ab28151) from Abcam (Cambridge, UK); cyclin-dependent kinase 1 (CDK1)-pT14 (1:1,000; cat. no. bs-3091R), cell division cycle 25C (CDC25C)-pS216 (1:1,000; cat. no. bs-3096R), β-actin (1:5,000; cat. no. bs-0061R) and matrix metalloproteinase 9 (MMP9; 1:1,000; cat. no. bs-4593R) from BIOSS].

Techniques: Western Blot, Incubation, SDS Page

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Analysis of apoptosis. (A) Representative images of nuclei stained with DAPI. HeLa cells were treated with 1× IC 50 or 2× IC 50 icaritin for 24 h (arrows point to nuclei of apoptotic cells). (B) Analysis of Annexin V-fluorescein isothiocyanate and propidium iodide-stained cells by flow cytometry. HeLa or SiHa cells were treated by 1× IC 50 or 2× IC 50 icaritin for 24 h or 48 h. (C) Quantification of flow cytometry data from three independent experiments. HeLa or SiHa cells with or without pre-incubation (1 h) with 5 mM NAC were treated with 1× IC 50 or 2× IC 50 icaritin for 24 h or 48 h (**P<0.01; ***P<0.001). (D) Measurement of mitochondrial membrane potential by JC-1. HeLa or SiHa cells were treated by 1× IC 50 or 2× IC 50 for 24 h. IC 50 , half maximal inhibitory concentration.

Article Snippet: The membranes were blocked in Tris-buffered saline with Tween-20 [10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween-20; PBST) containing 5% (w/v) nonfat milk for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C [B-cell lymphoma 2 (Bcl-2; 1:500; cat. no. sc-7382) and apoptosis regulator Bax (Bax; 1:500; cat. no. sc-7480) from Santa Cruz Biotechnology, Inc.; cleaved caspase 3 (1:500; cat. no. ab2302), cleaved caspase 9 (1:200; cat. no. ab2324) and X-linked inhibitor of apoptosis protein (XIAP; 1:1,000; cat. no. ab28151) from Abcam (Cambridge, UK); cyclin-dependent kinase 1 (CDK1)-pT14 (1:1,000; cat. no. bs-3091R), cell division cycle 25C (CDC25C)-pS216 (1:1,000; cat. no. bs-3096R), β-actin (1:5,000; cat. no. bs-0061R) and matrix metalloproteinase 9 (MMP9; 1:1,000; cat. no. bs-4593R) from BIOSS].

Techniques: Staining, Flow Cytometry, Incubation, Membrane, Concentration Assay

Journal: Oncology Reports

Article Title: Reactive oxygen species induced by icaritin promote DNA strand breaks and apoptosis in human cervical cancer cells

doi: 10.3892/or.2018.6864

Figure Lengend Snippet: Western blot analysis. HeLa and SiHa cells with or without pre-incubation (1 h) with 5 µM NAC were treated by icaritin for 24 h. Multiple SDS-PAGE gels were prepared for each sample, and each gel was used for the detection of one protein. Thus, one loading control, β-actin, was used for all proteins of each sample. (A) Detection of MMP-9. (B) Detection of cell cycle-associated proteins. (C) Detection of apoptosis-associated proteins. (D) Quantification of apoptosis-associated proteins in the 0 µM, 2× IC 50 and 2× IC 50 + NAC groups. Relative protein levels were quantified from western blots using ImageJ and presented as ratios of each protein band relative to the loading control. (*P<0.05, **P<0.01, ***P<0.001 vs. 0 µM). NAC, N-acetyl cysteine, MMP-9, matrix metalloproteinase-9; CDK1, cyclin-dependent kinase 1; CDC25C, cell division cycle 25C; XIAP, X-linked inhibitor of apoptosis protein; Bax, apoptosis regulator Bax; Bcl-2, B-cell lymphoma-2.

Article Snippet: The membranes were blocked in Tris-buffered saline with Tween-20 [10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween-20; PBST) containing 5% (w/v) nonfat milk for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C [B-cell lymphoma 2 (Bcl-2; 1:500; cat. no. sc-7382) and apoptosis regulator Bax (Bax; 1:500; cat. no. sc-7480) from Santa Cruz Biotechnology, Inc.; cleaved caspase 3 (1:500; cat. no. ab2302), cleaved caspase 9 (1:200; cat. no. ab2324) and X-linked inhibitor of apoptosis protein (XIAP; 1:1,000; cat. no. ab28151) from Abcam (Cambridge, UK); cyclin-dependent kinase 1 (CDK1)-pT14 (1:1,000; cat. no. bs-3091R), cell division cycle 25C (CDC25C)-pS216 (1:1,000; cat. no. bs-3096R), β-actin (1:5,000; cat. no. bs-0061R) and matrix metalloproteinase 9 (MMP9; 1:1,000; cat. no. bs-4593R) from BIOSS].

Techniques: Western Blot, Incubation, SDS Page, Control