Journal: Oncogene

Article Title: Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR.

doi: 10.1038/sj.onc.1203632

Figure Lengend Snippet: Figure 1 Immunohistochemical analysis for PKR expression in mammary carcinoma cell lines and nontransformed cell lines

Article Snippet: Six mammary carcinoma cells lines (MDA±MB435, MDA± MB468, T47D, MDA±MB231, MCF7 and MDA±MB453 from ATCC) and two nontransformed mammary epithelial cell lines (MCF10A and MCF12A from ATCC) were grown as monolayers in 10 cm plates as per the ATCC recommended protocol.

Techniques: Immunohistochemical staining, Expressing

Journal: Oncogene

Article Title: Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR.

doi: 10.1038/sj.onc.1203632

Figure Lengend Snippet: Figure 2 Activity of PKR is higher in mammary carcinoma cell lines than in nontransformed mammary epithelial cells. (A) PKR activation assay was performed using lysates (25 mg protein) of carcinoma (C) (lanes 1 ± 6) or nontransformed (N) (lanes 7, 8) mammary epithelial cell lines. PKR was immunoprecipitated and analysed by SDS ± polyacrylamide gel electrophoresis. (B) Quan- titation of the PKR activation assay. (C) Western blot analysis of lysates (25 mg protein) of carcinoma cell line, MCF7 (lane 2) or nontransformed cell line, MCF12A (lane 3) probed with PKR antibody speci®c for phospho-threonine258. Lane 1 contains puri®ed PKR autophosphorylated in vitro

Article Snippet: Six mammary carcinoma cells lines (MDA±MB435, MDA± MB468, T47D, MDA±MB231, MCF7 and MDA±MB453 from ATCC) and two nontransformed mammary epithelial cell lines (MCF10A and MCF12A from ATCC) were grown as monolayers in 10 cm plates as per the ATCC recommended protocol.

Techniques: Activity Assay, Activation Assay, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, In Vitro

Journal: Oncogene

Article Title: Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR.

doi: 10.1038/sj.onc.1203632

Figure Lengend Snippet: Figure 3 Phosphorylation of eIF2a and levels of eIF2B are higher in mammary carcinoma cell lines. (A) PKR activation assay was performed as described in Figure 1A in the presence of puri®ed human eIF2 using lysates of carcinoma (C) (lanes 1 ± 6) or nontransformed (N) (lanes 7, 8) cell lines and immunoprecipitated using both PKR antibody and eIF2a antibody and analysed as described in Materials and methods. (B) Phosphorylation of eIF2a by carcinoma cell lysate is dsRNA dependent. PKR assays with eIF2 was performed and analysed as described in (A) were performed in the presence (+) or absence (7) of dsRNA. Puri®ed PKR was used as a control (lane 5). (C) Endogenous eIF2a is phosphorylated in mammary carcinoma cell lines and not in nontransformed cell lines. Lysates from carcinoma (C) (lanes 3 ± 6) or nontransformed (N) (lanes 1, 2) cells were subjected to vertical gel isoelectric focusing electrophoresis, blotted onto an immobilon membrane and probed with eIF2a antibody as described in the Materials and methods. Positions of phosphorylated (eIF2aP) and nonphosphorylated eIF2a are marked. (D) Western blot analysis of lysates from carcinoma cells MCF7 or nontransformed cells MCF12A probed with either phospho-serine51 eIF2a antibody (top panel) or total eIF2a antibody (bottom panel). (E) Levels of eIF2B subunits are higher in carcinoma cell lines than in nontransformed cell lines. 50 mg protein from carcinoma cell lysates carcinoma (C) (lanes 4 ± 7) and nontransformed cell lysates (N) (lanes 2, 3) were run on a SDS ± polyacrylamide gel and subjected to Western blot analysis using antibodies to a or b subunits of eIF2B. Lane 1 contains puri®ed rabbit eIF2B as a positive control. The blot was also probed with actin antibody as a control for loading

Article Snippet: Six mammary carcinoma cells lines (MDA±MB435, MDA± MB468, T47D, MDA±MB231, MCF7 and MDA±MB453 from ATCC) and two nontransformed mammary epithelial cell lines (MCF10A and MCF12A from ATCC) were grown as monolayers in 10 cm plates as per the ATCC recommended protocol.

Techniques: Phospho-proteomics, Activation Assay, Immunoprecipitation, Control, Electrophoresis, Membrane, Western Blot, Positive Control

Journal: Oncogene

Article Title: Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR.

doi: 10.1038/sj.onc.1203632

Figure Lengend Snippet: Figure 4 Protein level of PKR in carcinoma cell lines is higher than in nontransformed cell lines (A) Lysates (25 mg protein) of carcinoma (C) (lanes 1 ± 4) or nontransformed (N) (lanes 5, 6) cells were subjected to Western blot analysis using PKR antibody. (B) Quantitation of the Western blot shown in (A). (C) PKR activity (shown in Figure 1b) normalized to PKR protein level in the cell lysates

Article Snippet: Six mammary carcinoma cells lines (MDA±MB435, MDA± MB468, T47D, MDA±MB231, MCF7 and MDA±MB453 from ATCC) and two nontransformed mammary epithelial cell lines (MCF10A and MCF12A from ATCC) were grown as monolayers in 10 cm plates as per the ATCC recommended protocol.

Techniques: Western Blot, Quantitation Assay, Activity Assay

Journal: Oncogene

Article Title: Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR.

doi: 10.1038/sj.onc.1203632

Figure Lengend Snippet: Figure 5 Nontransformed cell lines contain inhibitor of PKR activity. (A) PKR activation reactions with MCF7 cell lysate after preincubation alone (lane 1) or with increasing concentrations (5, 10, 25 and 50 mg) of MCF10A cell lysate (lanes 2 ± 5) were performed. Immunoprecipitation of PKR from the reactions and analysis were performed as described in Materials and methods. (B) PKR inhibitor in nontransformed cell lines was not a protease. PKR activation reaction using MCF7 cell lysate after preincubation alone (lane 1) or with MCF10A (lane 2) or with MCF12A (lane 3) cell lysates was subjected to Western blot analysis using PKR antibody. (C) Level of p58 is higher in nontransformed cell lines. Western blot analysis of lysates (50 mg) from carcinoma cell lines (C) and nontransformed cell lines (N) using p58 polyclonal antibody (upper panel) or actin monoclonal antibody (lower panel). (D) PKR inhibitor P58 interacts with PKR in nontransformed cells. Lysate (100 mg protein) of MCF12A (lane 1) or MCF7 (lane 3) or a mixture of MCF12A and MCF7 lysates (lane 2) was immunoprecipitated using anti-P58 and analysed for PKR by Western blot analysis using PKR monoclonal antibody. MCF12A lysate was also analysed using protein A-sepharose with no antibody as a negative control (lane 4). (E) Coimmunoprecipitation of radiolabeled PKR using P58 antibody from nontransformed cell lysate. Puri®ed PKR was radiolabeled in a kinase reaction in vitro, and mixed with lysate (100 mg protein) of MCF12A (lane 2) or MCF7 (lane 3) before immunoprecipitating with anti-P58 bound protein A-sepharose as described in Materials and methods. Lane 1 contains 20% of the radiolabeled PKR that was mixed with the lysates before immunoprecipitation. Lanes 4 and 5 are no lysate and no antibody negative controls, respectively

Article Snippet: Six mammary carcinoma cells lines (MDA±MB435, MDA± MB468, T47D, MDA±MB231, MCF7 and MDA±MB453 from ATCC) and two nontransformed mammary epithelial cell lines (MCF10A and MCF12A from ATCC) were grown as monolayers in 10 cm plates as per the ATCC recommended protocol.

Techniques: Activity Assay, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, In Vitro

Journal: Cancer medicine

Article Title: Arid4a Suppresses Breast Tumor Metastasis by Enhancing MTSS1 Expression via mRNA Stability.

doi: 10.1002/cam4.70732

Figure Lengend Snippet: FIGURE 1 | Arid4a is expressed at relatively low levels in breast tumors and indicates poor prognosis. (A) The expression of Arid4a between nor- mal breast tissues and primary breast tumors was analyzed (http://ualcan.path.uab.edu/analysis-prot.html). (B) Arid4a expression was measured by qPCR in human breast tumors and surrounding healthy tissues (n = 16). (C) Comparison of Arid4a expression between different subtypes of breast cancers (http://ualcan.path.uab.edu/analysis-prot.html). (D) Arid4a protein expression in human primary breast tumors and normal tissues was analyzed (http://ualcan.path.uab.edu/analysis-prot.html). (E) Left: Representative images of Arid4a IHC stating in normal mammary gland tissues and breast tumor tissues were shown. Scale bar, 100 μm. Right: IHC scores of Arid4a staining. (F) Arid4a protein levels were measured by immunoblotting in human normal mammary gland epithelial cell lines (MCF-10A and MCF-12A) and breast tumor cell lines (MDA-MB-231, MCF7, MDA-MB-468, and T47D). (G) Arid4a expression was analyzed based on nodal metastasis status of breast cancers (http://ualcan.path.uab.edu/ analysis-prot.html). (H) Kaplan–Meier overall survival and relapse free survival curves of breast cancer patients with low and high tumor Arid4a transcripts (kmplot.com). (I) Kaplan–Meier distant metastasis free survival (DMFS), disease free survival (DFS), relapse free survival, and disease specific survival (DSS) curves of breast cancer patients with low and high tumor Arid4a transcripts (PrognoScan: A new database for meta-analysis of the prognostic value of genes. (kyutech.ac.jp)). *p < 0.05; ****p < 0.00001.

Article Snippet: Human breast cancer cell lines (MDA- MB- 231, MDA- MB- 468, MCF7 and T47D) and human normal mammary epithelial cell lines (MCF- 10A and MCF- 12A) were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Comparison, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The Antioxidant Transcription Factor Nrf2 Negatively Regulates Autophagy and Growth Arrest Induced by the Anticancer Redox Agent Mitoquinone *

doi: 10.1074/jbc.M110.133579

Figure Lengend Snippet: Growth inhibition and cell cycle arrest by MitoQ or CoQ10. A and B, an SRB dye-based assay was used for measuring cell viability following increasing concentrations of either MitoQ (left) or CoQ10 (right) after 72 h. Breast cancer cell lines (MDA-MB-231 or MCF-7) or healthy breast epithelial cells (MCF-12A) were treated as indicated. B, a colony formation assay was performed on MDA-MB-231 cells incubated with MitoQ. Cells were incubated with MitoQ for 6 h, followed by replacement of medium and replating for a colony formation period of 7 days. C, MDA-MB-231 cells were treated with either MitoQ or CoQ10, as indicated, for 48 h. The cells were fixed and stained with PI followed by flow cytometric analysis. The percentage of cells in each cell cycle phase was quantitated and is provided in each panel. D, the phosphorylation of Chk1 and Chk2 kinases was assayed using confocal microscopy. Representative images of increased nuclear foci corresponding to phospho-Chk1-Ser317 (pChk1-S317), phospho-Chk2-Thr68 (pChk1-T68), and cytoplasmic phospho-Chk1-Ser345 (pChk1-S345) after 0.1 μm MitoQ or 0.1 μm doxorubicin (Dox) treatment (24 h) are shown. Error bars, S.D.

Article Snippet: MCF-12A healthy human mammary epithelial cells were obtained from BioWhittaker Clonetics (Walkersville, MD) and maintained in mammary epithelial cell growth medium ( 16 ).

Techniques: Inhibition, Colony Assay, Incubation, Staining, Confocal Microscopy