Journal: Scientific Reports

Article Title: HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction

doi: 10.1038/srep07201

Figure Lengend Snippet: (A) Overexpression of HER2CA increases the NRF2-induced expressions of HO-1 and MRP5 in MCF7 cells. MCF7 cells were transiently transfected with expression vectors by Lipofectamine PLUS as indicated and subjected to western blot analysis. β-actin was used as a loading control. The blots used in this experiment was cut into several pieces according to estimated M.W. of proteins of interest and probed indicated antibodies. Different pieces of the same protein blots or same samples with same conditions of electrophoresis and electrotransfer were used and presented. (B) HER2CA increases NRF2 DNA-binding in MCF7 cells. MCF7 cells were transfected with expression vectors as indicated and NRF2 DNA-binding assay was performed. As a control, MCF7 cells were treated with 100 µM of tBHQ for 8 hr. (C) HER2CA increases the ARE-Luc reporter gene activity in an NRF2-depedent manner. MCF7 cells were transfected with expression vectors as indicated with Lipofectamine PLUS and luciferase activity was determined as described in Materials and methods. (D) HER2CA increased transcriptional activation of HO-1 promoter by endogenous NRF2. MCF7 cells were transfected with HO-1 promoter-Luc and increasing amounts of HER2CA expression vector in the absence or presence of KEAP1 knockdown. Luciferase assay were performed as described in (C). (E) HER2CA increases the NRF2-dependent transcription of HO-1 promoter. MCF7 cells transfected with expression vectors by Lipofectamine PLUS as indicated in the presence of HO-1 promoter-Luc containing either wild type or mutant AP-1 site. Luciferase activity was determined as in (B). (B,C) Representative data were presented as mean ± SEM of triplicate experiments. Statistically significance was determined by two-tailed Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; and *** P ≤ 0.001.

Article Snippet: Antibodies used in this study were purchased from the following sources: phospho-AKT (S473) (#9271), AKT (#9272), phospho-ERK1/2 (T202/Y204) (#4370), and HER2 (#2247) from Cell Signaling Technologies, Inc. (Danvers, MA); HO-1 (ADI-OSA-110) from Enzo Life Sciences (Farmingdale, NY); β-actin, ERK1 (sc-94), NRF2 (sc-13032), and MRP5 (sc-5770) from Santa Cruz Biotechnology (Santa Cruz, CA); FLAG M2 (F1804) from Sigma; and GFP (ab290-50) from Abcam (Cambridge, MA).

Techniques: Over Expression, Transfection, Expressing, Western Blot, Control, Electrophoresis, Electrotransfer, Binding Assay, DNA Binding Assay, Activity Assay, Luciferase, Activation Assay, Plasmid Preparation, Knockdown, Mutagenesis, Two Tailed Test

Journal: Scientific Reports

Article Title: HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction

doi: 10.1038/srep07201

Figure Lengend Snippet: (A) RT-qPCR analysis of HER2CA and FLAG-NRF2 expression in MCF7 cells after transient transfection of expression vectors by Lipofectamine PLUS as indicated. Transfection was performed as in . (B–F) RT-qPCR analysis of multiple gene expressions. (G) Schematic diagram of the proposed regulation of gene expression by HER2 and NRF2. (A–F) Representative data are shown as mean ± SEM of quadruplicate experiments. Statistically significance was determined by two-tailed Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; and *** P ≤ 0.001.

Article Snippet: Antibodies used in this study were purchased from the following sources: phospho-AKT (S473) (#9271), AKT (#9272), phospho-ERK1/2 (T202/Y204) (#4370), and HER2 (#2247) from Cell Signaling Technologies, Inc. (Danvers, MA); HO-1 (ADI-OSA-110) from Enzo Life Sciences (Farmingdale, NY); β-actin, ERK1 (sc-94), NRF2 (sc-13032), and MRP5 (sc-5770) from Santa Cruz Biotechnology (Santa Cruz, CA); FLAG M2 (F1804) from Sigma; and GFP (ab290-50) from Abcam (Cambridge, MA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Gene Expression, Two Tailed Test

Journal: Scientific Reports

Article Title: HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction

doi: 10.1038/srep07201

Figure Lengend Snippet: (A,B) HEK293T cells were transfected with HER2CA and (A) FLAG-NRF2 or (B) GFP-NRF2 expression vectors as indicated. (C) HER2 binds to NRF2 in HER2-amplified breast cancer cells. Cells were serum-starved for 24 hr and stimulated by 100 ng/ml of HRG for 4 hr before cell lysis. (A–C) The blots used in this experiment was cut into couples of pieces according to estimated M.W. of proteins of interest and probed with indicated antibodies. Discontinuous lanes from same blots were clearly marked as shown. Immunoprecipitation and western blot analysis were performed with indicated antibodies. β-actin was used as a loading control.

Article Snippet: Antibodies used in this study were purchased from the following sources: phospho-AKT (S473) (#9271), AKT (#9272), phospho-ERK1/2 (T202/Y204) (#4370), and HER2 (#2247) from Cell Signaling Technologies, Inc. (Danvers, MA); HO-1 (ADI-OSA-110) from Enzo Life Sciences (Farmingdale, NY); β-actin, ERK1 (sc-94), NRF2 (sc-13032), and MRP5 (sc-5770) from Santa Cruz Biotechnology (Santa Cruz, CA); FLAG M2 (F1804) from Sigma; and GFP (ab290-50) from Abcam (Cambridge, MA).

Techniques: Transfection, Expressing, Amplification, Lysis, Immunoprecipitation, Western Blot, Control

Journal: Scientific Reports

Article Title: HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction

doi: 10.1038/srep07201

Figure Lengend Snippet: (A) Overexpression of HER2CA increases the NRF2-induced expressions of HO-1 and MRP5 in MCF7 cells. MCF7 cells were transiently transfected with expression vectors by Lipofectamine PLUS as indicated and subjected to western blot analysis. β-actin was used as a loading control. The blots used in this experiment was cut into several pieces according to estimated M.W. of proteins of interest and probed indicated antibodies. Different pieces of the same protein blots or same samples with same conditions of electrophoresis and electrotransfer were used and presented. (B) HER2CA increases NRF2 DNA-binding in MCF7 cells. MCF7 cells were transfected with expression vectors as indicated and NRF2 DNA-binding assay was performed. As a control, MCF7 cells were treated with 100 µM of tBHQ for 8 hr. (C) HER2CA increases the ARE-Luc reporter gene activity in an NRF2-depedent manner. MCF7 cells were transfected with expression vectors as indicated with Lipofectamine PLUS and luciferase activity was determined as described in Materials and methods. (D) HER2CA increased transcriptional activation of HO-1 promoter by endogenous NRF2. MCF7 cells were transfected with HO-1 promoter-Luc and increasing amounts of HER2CA expression vector in the absence or presence of KEAP1 knockdown. Luciferase assay were performed as described in (C). (E) HER2CA increases the NRF2-dependent transcription of HO-1 promoter. MCF7 cells transfected with expression vectors by Lipofectamine PLUS as indicated in the presence of HO-1 promoter-Luc containing either wild type or mutant AP-1 site. Luciferase activity was determined as in (B). (B,C) Representative data were presented as mean ± SEM of triplicate experiments. Statistically significance was determined by two-tailed Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; and *** P ≤ 0.001.

Article Snippet: Antibodies used in this study were purchased from the following sources: phospho-AKT (S473) (#9271), AKT (#9272), phospho-ERK1/2 (T202/Y204) (#4370), and HER2 (#2247) from Cell Signaling Technologies, Inc. (Danvers, MA); HO-1 (ADI-OSA-110) from Enzo Life Sciences (Farmingdale, NY); β-actin, ERK1 (sc-94), NRF2 (sc-13032), and MRP5 (sc-5770) from Santa Cruz Biotechnology (Santa Cruz, CA); FLAG M2 (F1804) from Sigma; and GFP (ab290-50) from Abcam (Cambridge, MA).

Techniques: Over Expression, Transfection, Expressing, Western Blot, Control, Electrophoresis, Electrotransfer, Binding Assay, DNA Binding Assay, Activity Assay, Luciferase, Activation Assay, Plasmid Preparation, Knockdown, Mutagenesis, Two Tailed Test

Journal: Scientific Reports

Article Title: HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction

doi: 10.1038/srep07201

Figure Lengend Snippet: (A,B) HEK293T cells were transfected with HER2CA and (A) FLAG-NRF2 or (B) GFP-NRF2 expression vectors as indicated. (C) HER2 binds to NRF2 in HER2-amplified breast cancer cells. Cells were serum-starved for 24 hr and stimulated by 100 ng/ml of HRG for 4 hr before cell lysis. (A–C) The blots used in this experiment was cut into couples of pieces according to estimated M.W. of proteins of interest and probed with indicated antibodies. Discontinuous lanes from same blots were clearly marked as shown. Immunoprecipitation and western blot analysis were performed with indicated antibodies. β-actin was used as a loading control.

Article Snippet: Antibodies used in this study were purchased from the following sources: phospho-AKT (S473) (#9271), AKT (#9272), phospho-ERK1/2 (T202/Y204) (#4370), and HER2 (#2247) from Cell Signaling Technologies, Inc. (Danvers, MA); HO-1 (ADI-OSA-110) from Enzo Life Sciences (Farmingdale, NY); β-actin, ERK1 (sc-94), NRF2 (sc-13032), and MRP5 (sc-5770) from Santa Cruz Biotechnology (Santa Cruz, CA); FLAG M2 (F1804) from Sigma; and GFP (ab290-50) from Abcam (Cambridge, MA).

Techniques: Transfection, Expressing, Amplification, Lysis, Immunoprecipitation, Western Blot, Control