mouse erbb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse erbb2
    A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to <t>ERBB2</t> was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.
    Mouse Erbb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies"

    Article Title: Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112376

    A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to ERBB2 was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.
    Figure Legend Snippet: A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to ERBB2 was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.

    Techniques Used: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Standard Deviation, Incubation, Flow Cytometry, Positive Control, Negative Control

    Individual, pairwise, and triple combinations of A5, F4 and either trastuzumab or erlotinib (as appropriate) were serially diluted (2-fold dilution scheme) and incubated with the ERBB2-positive cell lines, BT-474, SK-BR-3, NCI-N87, and EGFR-positive cell line ACHN, as noted. Concentration plotted on the x-axis refers to anti-ERBB3 IgG concentration. Trastuzumab and erlotinib are at 1∶200 molar ratios with anti-ERBB3 antibodies. The impact of treatment on cell viability was quantified with Cell-Titer Blue. Vehicle-treated controls for each cell line were set at 100%. Values plotted represent the means ± standard error of the means (SEM) for between 2–6 independent experiments, each carried out in triplicate.
    Figure Legend Snippet: Individual, pairwise, and triple combinations of A5, F4 and either trastuzumab or erlotinib (as appropriate) were serially diluted (2-fold dilution scheme) and incubated with the ERBB2-positive cell lines, BT-474, SK-BR-3, NCI-N87, and EGFR-positive cell line ACHN, as noted. Concentration plotted on the x-axis refers to anti-ERBB3 IgG concentration. Trastuzumab and erlotinib are at 1∶200 molar ratios with anti-ERBB3 antibodies. The impact of treatment on cell viability was quantified with Cell-Titer Blue. Vehicle-treated controls for each cell line were set at 100%. Values plotted represent the means ± standard error of the means (SEM) for between 2–6 independent experiments, each carried out in triplicate.

    Techniques Used: Incubation, Concentration Assay

    mouse erbb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse erbb2
    A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to <t>ERBB2</t> was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.
    Mouse Erbb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies"

    Article Title: Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112376

    A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to ERBB2 was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.
    Figure Legend Snippet: A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to ERBB2 was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.

    Techniques Used: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Standard Deviation, Incubation, Flow Cytometry, Positive Control, Negative Control

    Individual, pairwise, and triple combinations of A5, F4 and either trastuzumab or erlotinib (as appropriate) were serially diluted (2-fold dilution scheme) and incubated with the ERBB2-positive cell lines, BT-474, SK-BR-3, NCI-N87, and EGFR-positive cell line ACHN, as noted. Concentration plotted on the x-axis refers to anti-ERBB3 IgG concentration. Trastuzumab and erlotinib are at 1∶200 molar ratios with anti-ERBB3 antibodies. The impact of treatment on cell viability was quantified with Cell-Titer Blue. Vehicle-treated controls for each cell line were set at 100%. Values plotted represent the means ± standard error of the means (SEM) for between 2–6 independent experiments, each carried out in triplicate.
    Figure Legend Snippet: Individual, pairwise, and triple combinations of A5, F4 and either trastuzumab or erlotinib (as appropriate) were serially diluted (2-fold dilution scheme) and incubated with the ERBB2-positive cell lines, BT-474, SK-BR-3, NCI-N87, and EGFR-positive cell line ACHN, as noted. Concentration plotted on the x-axis refers to anti-ERBB3 IgG concentration. Trastuzumab and erlotinib are at 1∶200 molar ratios with anti-ERBB3 antibodies. The impact of treatment on cell viability was quantified with Cell-Titer Blue. Vehicle-treated controls for each cell line were set at 100%. Values plotted represent the means ± standard error of the means (SEM) for between 2–6 independent experiments, each carried out in triplicate.

    Techniques Used: Incubation, Concentration Assay

    her2 cst  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc her2 cst
    Zotatifin translationally downregulates FGFR1/2 and <t>HER2</t> in RTK-driven tumors. (A) Schematic of RTK-stimulated PI3K/AKT and RAS/ERK pathways that activate eIF4A. (B) Frequency of HER2 and FGFR1/2 dysregulation in different cancer types. (C) Western blot analysis of RTKs and cyclin D1 levels following 24 h treatment with zotatifin or DMSO in HER2- or FGFR1/2-driven tumor models. β-actin or α-tubulin serve as loading controls. (D) Polysome profiling and mRNA distribution in MDA-MB-361 breast cancer cells treated for 3 h with 20 nM zotatifin compared to DMSO. mRNA levels of HER2 are monitored in polysome fractions. GAPDH serves as a control. (E) Luciferase reporter assay with gene constructs containing 5’-UTRs of FGFR1/2, HER2 or TUBA in HEK293 cells following 4 h of dose-dependent treatment with zotatifin. (F) Left, list of genes of which their 5’-UTR sequences were cloned into luciferase reporter constructs. Right, measured IC 50 values (nM) for inhibition of expression following 4 h treatment with zotatifin.
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    1) Product Images from "Zotatifin, an eIF4A-Selective Inhibitor, Blocks Tumor Growth in Receptor Tyrosine Kinase Driven Tumors"

    Article Title: Zotatifin, an eIF4A-Selective Inhibitor, Blocks Tumor Growth in Receptor Tyrosine Kinase Driven Tumors

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2021.766298

    Zotatifin translationally downregulates FGFR1/2 and HER2 in RTK-driven tumors. (A) Schematic of RTK-stimulated PI3K/AKT and RAS/ERK pathways that activate eIF4A. (B) Frequency of HER2 and FGFR1/2 dysregulation in different cancer types. (C) Western blot analysis of RTKs and cyclin D1 levels following 24 h treatment with zotatifin or DMSO in HER2- or FGFR1/2-driven tumor models. β-actin or α-tubulin serve as loading controls. (D) Polysome profiling and mRNA distribution in MDA-MB-361 breast cancer cells treated for 3 h with 20 nM zotatifin compared to DMSO. mRNA levels of HER2 are monitored in polysome fractions. GAPDH serves as a control. (E) Luciferase reporter assay with gene constructs containing 5’-UTRs of FGFR1/2, HER2 or TUBA in HEK293 cells following 4 h of dose-dependent treatment with zotatifin. (F) Left, list of genes of which their 5’-UTR sequences were cloned into luciferase reporter constructs. Right, measured IC 50 values (nM) for inhibition of expression following 4 h treatment with zotatifin.
    Figure Legend Snippet: Zotatifin translationally downregulates FGFR1/2 and HER2 in RTK-driven tumors. (A) Schematic of RTK-stimulated PI3K/AKT and RAS/ERK pathways that activate eIF4A. (B) Frequency of HER2 and FGFR1/2 dysregulation in different cancer types. (C) Western blot analysis of RTKs and cyclin D1 levels following 24 h treatment with zotatifin or DMSO in HER2- or FGFR1/2-driven tumor models. β-actin or α-tubulin serve as loading controls. (D) Polysome profiling and mRNA distribution in MDA-MB-361 breast cancer cells treated for 3 h with 20 nM zotatifin compared to DMSO. mRNA levels of HER2 are monitored in polysome fractions. GAPDH serves as a control. (E) Luciferase reporter assay with gene constructs containing 5’-UTRs of FGFR1/2, HER2 or TUBA in HEK293 cells following 4 h of dose-dependent treatment with zotatifin. (F) Left, list of genes of which their 5’-UTR sequences were cloned into luciferase reporter constructs. Right, measured IC 50 values (nM) for inhibition of expression following 4 h treatment with zotatifin.

    Techniques Used: Western Blot, Luciferase, Reporter Assay, Construct, Clone Assay, Inhibition, Expressing

    Zotatifin downregulates RTK levels induced by PI3K/AKT inhibition feedback in vitro . (A, B) SK-BR-3 HER2 amp cell line treated with zotatifin and Ipatasertib (AKTi); (A) Proliferation and apoptosis induction curves following 72 h of single agent or combination treatments (see also <xref ref-type= Figures 4B, C ). (B) Western blots analysis following 48 h treatment with single agents or combinations. (C) JIMT-1 HER2 amp cell line treated with zotatifin and Alpelisib (PIK3CAi) for 24 h; (D) MFM-223 FGFR2 amp cell line treated with zotatifin and Ipatasertib (AKTi) for 24 h; β-actin or GAPDH serve as loading controls. Quantification of p-HER3 Y1289 protein levels is shown for each condition. Concentrations of drugs used are indicated on top of each blot: Ipa., Ipatasertib (µM); Alp., Alpelisib (µM); zotatifin (nM); D, DMSO. " title="... inhibition feedback in vitro . (A, B) SK-BR-3 HER2 amp cell line treated with zotatifin and Ipatasertib ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Zotatifin downregulates RTK levels induced by PI3K/AKT inhibition feedback in vitro . (A, B) SK-BR-3 HER2 amp cell line treated with zotatifin and Ipatasertib (AKTi); (A) Proliferation and apoptosis induction curves following 72 h of single agent or combination treatments (see also Figures 4B, C ). (B) Western blots analysis following 48 h treatment with single agents or combinations. (C) JIMT-1 HER2 amp cell line treated with zotatifin and Alpelisib (PIK3CAi) for 24 h; (D) MFM-223 FGFR2 amp cell line treated with zotatifin and Ipatasertib (AKTi) for 24 h; β-actin or GAPDH serve as loading controls. Quantification of p-HER3 Y1289 protein levels is shown for each condition. Concentrations of drugs used are indicated on top of each blot: Ipa., Ipatasertib (µM); Alp., Alpelisib (µM); zotatifin (nM); D, DMSO.

    Techniques Used: Inhibition, In Vitro, Western Blot

    anti her2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti her2
    TRIB3 and <t>HER2</t> expression exhibit a positive correlation in breast cancer. ( a ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in a panel of breast cancer cell lines (RNA seq data were obtained from the GSE48216 dataset). ( n = 56; p and R were calculated using Spearman’s correlation coefficient). ( b ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in breast cancer patients (RNA seq data were obtained from the TGCA Breast cancer dataset using the UCSC Xena platform) ( n = 1247; p and R were calculated using the Spearman correlation coefficient). ( c ) Correlation between TRIB3 protein levels (as determined by TRIB3 immunostaining and the subsequent assignment of the corresponding H value) with respect to HER2 levels (pathologically classified as 0-1-2-3 according to the level of expression of this receptor) in a tissue microarray that was generated with samples that were derived from luminal breast cancer patients ( n = 159; p was calculated using the Spearman’s correlation coefficient).
    Anti Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Pseudokinase TRIB3 Negatively Regulates the HER2 Receptor Pathway and Is a Biomarker of Good Prognosis in Luminal Breast Cancer"

    Article Title: The Pseudokinase TRIB3 Negatively Regulates the HER2 Receptor Pathway and Is a Biomarker of Good Prognosis in Luminal Breast Cancer

    Journal: Cancers

    doi: 10.3390/cancers13215307

    TRIB3 and HER2 expression exhibit a positive correlation in breast cancer. ( a ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in a panel of breast cancer cell lines (RNA seq data were obtained from the GSE48216 dataset). ( n = 56; p and R were calculated using Spearman’s correlation coefficient). ( b ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in breast cancer patients (RNA seq data were obtained from the TGCA Breast cancer dataset using the UCSC Xena platform) ( n = 1247; p and R were calculated using the Spearman correlation coefficient). ( c ) Correlation between TRIB3 protein levels (as determined by TRIB3 immunostaining and the subsequent assignment of the corresponding H value) with respect to HER2 levels (pathologically classified as 0-1-2-3 according to the level of expression of this receptor) in a tissue microarray that was generated with samples that were derived from luminal breast cancer patients ( n = 159; p was calculated using the Spearman’s correlation coefficient).
    Figure Legend Snippet: TRIB3 and HER2 expression exhibit a positive correlation in breast cancer. ( a ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in a panel of breast cancer cell lines (RNA seq data were obtained from the GSE48216 dataset). ( n = 56; p and R were calculated using Spearman’s correlation coefficient). ( b ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in breast cancer patients (RNA seq data were obtained from the TGCA Breast cancer dataset using the UCSC Xena platform) ( n = 1247; p and R were calculated using the Spearman correlation coefficient). ( c ) Correlation between TRIB3 protein levels (as determined by TRIB3 immunostaining and the subsequent assignment of the corresponding H value) with respect to HER2 levels (pathologically classified as 0-1-2-3 according to the level of expression of this receptor) in a tissue microarray that was generated with samples that were derived from luminal breast cancer patients ( n = 159; p was calculated using the Spearman’s correlation coefficient).

    Techniques Used: Expressing, RNA Sequencing Assay, Immunostaining, Microarray, Generated, Derivative Assay

    TRIB3 and HER2 mutually regulate their levels. ( a ) The effect of HER2 silencing (siHER2) or transfection with a control siRNA (siC) on TRIB3 levels of BT474 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to siC ( n = 5; * p < 0.05 by using the Wilcoxon test). ( b ) The effect of HER2 overexpression (+HER2) or transfection with an empty vector (Φ) on TRIB3 levels of MCF7 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to the empty vector-transfected cells (Φ) ( n = 5; * p < 0.05 by using the Wilcoxon test). ( c ) Effect of TRIB3 silencing on HER2 mRNA (as determined by qRT-PCR) levels of BT474 cells. The data correspond to HER2 mRNA normalized with respect to the loading control (GAPDH) and are expressed as the mean fold change ± SEM with respect the shC condition ( n = 3). ( d ) The effect of TRIB3 stable knock-down on HER2 protein (as determined by Western blot) of MDA-MB-361, AU565, and BT474 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (β-ACTIN) and are expressed as the mean fold change ± SEM with respect to the shC condition ( n = 7; * p < 0.05 by using the Wilcoxon test). ( e ) The effect of TRIB3 silencing and incubation with the protein synthesis inhibitor cycloheximide (10 µM, CHX) on HER2 protein levels of BT474 cells at different timepoints. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to 0 h timepoint ( n = 3; * p < 0.05 with respect to shC cells; using the two-way ANOVA test). ( f ) The effect of treatment with Lapatinib (4 μM, 48 h) or vehicle (veh, DMSO) on the number of BT474 shC and shTRIB3 cells (as estimated by crystal violet staining). The data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 5; * p < 0.05 with respect to shC cells; using the two-way ANOVA test). ( g ) The effect of treatment with the AKT X inhibitor (10 μM, 48 h) or vehicle (veh, H 2 O) on the number of BT474 shC and shTRIB3 cells (as estimated by crystal violet staining). Data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 6; * p < 0.05, with respect to the shC cells; using the two-way ANOVA test).
    Figure Legend Snippet: TRIB3 and HER2 mutually regulate their levels. ( a ) The effect of HER2 silencing (siHER2) or transfection with a control siRNA (siC) on TRIB3 levels of BT474 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to siC ( n = 5; * p < 0.05 by using the Wilcoxon test). ( b ) The effect of HER2 overexpression (+HER2) or transfection with an empty vector (Φ) on TRIB3 levels of MCF7 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to the empty vector-transfected cells (Φ) ( n = 5; * p < 0.05 by using the Wilcoxon test). ( c ) Effect of TRIB3 silencing on HER2 mRNA (as determined by qRT-PCR) levels of BT474 cells. The data correspond to HER2 mRNA normalized with respect to the loading control (GAPDH) and are expressed as the mean fold change ± SEM with respect the shC condition ( n = 3). ( d ) The effect of TRIB3 stable knock-down on HER2 protein (as determined by Western blot) of MDA-MB-361, AU565, and BT474 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (β-ACTIN) and are expressed as the mean fold change ± SEM with respect to the shC condition ( n = 7; * p < 0.05 by using the Wilcoxon test). ( e ) The effect of TRIB3 silencing and incubation with the protein synthesis inhibitor cycloheximide (10 µM, CHX) on HER2 protein levels of BT474 cells at different timepoints. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to 0 h timepoint ( n = 3; * p < 0.05 with respect to shC cells; using the two-way ANOVA test). ( f ) The effect of treatment with Lapatinib (4 μM, 48 h) or vehicle (veh, DMSO) on the number of BT474 shC and shTRIB3 cells (as estimated by crystal violet staining). The data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 5; * p < 0.05 with respect to shC cells; using the two-way ANOVA test). ( g ) The effect of treatment with the AKT X inhibitor (10 μM, 48 h) or vehicle (veh, H 2 O) on the number of BT474 shC and shTRIB3 cells (as estimated by crystal violet staining). Data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 6; * p < 0.05, with respect to the shC cells; using the two-way ANOVA test).

    Techniques Used: Transfection, Western Blot, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Incubation, Staining

    High TRIB3 levels correlate with better response to therapy in both HER2- and HER2+ luminal breast cancer. ( a ) The bioinformatic analysis of the association between TRIB3 mRNA levels and complete pathological response to endocrine therapies in ER-positive luminal A (left panel) and luminal B (right panel) breast cancer patients. The receiver operating characteristic (ROC) curves are shown. The area under the curve (AUC), p value, false positive rate (FPR), and true positive rate (TPR). ( b , c ) The correlation between TRIB3 protein levels (as determined by TRIB3 immunostaining) and recurrence after treatment with chemotherapy in a TMA of luminal breast cancer patients. Note that all patients had been initially treated with endocrine therapy. The data correspond to the H-value quantification of TRIB3 levels in the primary tumors and are expressed as the mean H value ± SEM for each set of patients. Panels b and c show information relative to all of the patients that were subjected to chemotherapy irrespective of HER2 status (panel B, n = 150) or separated according to HER2 expression (Panel c, n = 32 for HER2+ and n = 118 for HER2-). ** p < 0.01 and * p < 0.05 with respect to non-recurrent patients by using a T-test with Welch correction. ( d – g ) The effect of TRIB3 silencing (shTRIB3) and treatment with Doxorubicin ( d , e ) or Cisplatin ( f , g ) on the number of BT474 ( e , f ) and MCF7 ( d , g ) cells (as estimated by crystal violet staining). The data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 3–5; ** p < 0.01 and * p < 0.05 with respect to shC cells; using the two-way ANOVA test).
    Figure Legend Snippet: High TRIB3 levels correlate with better response to therapy in both HER2- and HER2+ luminal breast cancer. ( a ) The bioinformatic analysis of the association between TRIB3 mRNA levels and complete pathological response to endocrine therapies in ER-positive luminal A (left panel) and luminal B (right panel) breast cancer patients. The receiver operating characteristic (ROC) curves are shown. The area under the curve (AUC), p value, false positive rate (FPR), and true positive rate (TPR). ( b , c ) The correlation between TRIB3 protein levels (as determined by TRIB3 immunostaining) and recurrence after treatment with chemotherapy in a TMA of luminal breast cancer patients. Note that all patients had been initially treated with endocrine therapy. The data correspond to the H-value quantification of TRIB3 levels in the primary tumors and are expressed as the mean H value ± SEM for each set of patients. Panels b and c show information relative to all of the patients that were subjected to chemotherapy irrespective of HER2 status (panel B, n = 150) or separated according to HER2 expression (Panel c, n = 32 for HER2+ and n = 118 for HER2-). ** p < 0.01 and * p < 0.05 with respect to non-recurrent patients by using a T-test with Welch correction. ( d – g ) The effect of TRIB3 silencing (shTRIB3) and treatment with Doxorubicin ( d , e ) or Cisplatin ( f , g ) on the number of BT474 ( e , f ) and MCF7 ( d , g ) cells (as estimated by crystal violet staining). The data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 3–5; ** p < 0.01 and * p < 0.05 with respect to shC cells; using the two-way ANOVA test).

    Techniques Used: Immunostaining, Expressing, Staining

    her2  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc her2
    A BT474 cells were treated with Leptin and AG1478 alone or in combination for 24 h. Cell lysates were immunoblotted with indicated antibodies. B BT474 cells were treated with Leptin and AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMed1, pHER2, and tHER2. Actin was used as loading control. C Bar graph shows fold change in protein expression in ( A ) and ( B ). D Immunoblot analysis of MCF7-vector and <t>MCF7-Her2</t> O/E cells for indicated proteins. E , F MCF7-HER2 O/E cells were treated with Leptin, AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMED1, pHER2, and tHER2. Actin was used as loading control. Bar graph shows the ratio of MED1 and actin. G MCF7 and BT474 cells were treated as in ( A ) and ( B ), and subjected to immunofluorescence analysis of pMED1. Scale bar, 10 µm. H MCF7 cells were treated as indicated for 24 h and subjected to ChIP assay using Med1 antibody. The purified DNA was analyzed by using specific primer for ER promoter. ** p < 0.005, * p < 0.05. Data are means ± SD from three experiments.
    Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen by coordinating a crosstalk between Med1, miR205 and ErbB"

    Article Title: Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen by coordinating a crosstalk between Med1, miR205 and ErbB

    Journal: NPJ Breast Cancer

    doi: 10.1038/s41523-021-00314-9

    A BT474 cells were treated with Leptin and AG1478 alone or in combination for 24 h. Cell lysates were immunoblotted with indicated antibodies. B BT474 cells were treated with Leptin and AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMed1, pHER2, and tHER2. Actin was used as loading control. C Bar graph shows fold change in protein expression in ( A ) and ( B ). D Immunoblot analysis of MCF7-vector and MCF7-Her2 O/E cells for indicated proteins. E , F MCF7-HER2 O/E cells were treated with Leptin, AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMED1, pHER2, and tHER2. Actin was used as loading control. Bar graph shows the ratio of MED1 and actin. G MCF7 and BT474 cells were treated as in ( A ) and ( B ), and subjected to immunofluorescence analysis of pMED1. Scale bar, 10 µm. H MCF7 cells were treated as indicated for 24 h and subjected to ChIP assay using Med1 antibody. The purified DNA was analyzed by using specific primer for ER promoter. ** p < 0.005, * p < 0.05. Data are means ± SD from three experiments.
    Figure Legend Snippet: A BT474 cells were treated with Leptin and AG1478 alone or in combination for 24 h. Cell lysates were immunoblotted with indicated antibodies. B BT474 cells were treated with Leptin and AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMed1, pHER2, and tHER2. Actin was used as loading control. C Bar graph shows fold change in protein expression in ( A ) and ( B ). D Immunoblot analysis of MCF7-vector and MCF7-Her2 O/E cells for indicated proteins. E , F MCF7-HER2 O/E cells were treated with Leptin, AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMED1, pHER2, and tHER2. Actin was used as loading control. Bar graph shows the ratio of MED1 and actin. G MCF7 and BT474 cells were treated as in ( A ) and ( B ), and subjected to immunofluorescence analysis of pMED1. Scale bar, 10 µm. H MCF7 cells were treated as indicated for 24 h and subjected to ChIP assay using Med1 antibody. The purified DNA was analyzed by using specific primer for ER promoter. ** p < 0.005, * p < 0.05. Data are means ± SD from three experiments.

    Techniques Used: Expressing, Western Blot, Plasmid Preparation, Immunofluorescence, Purification

    her2 erbb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc her2 erbb2
    A Western blot and quantification of the levels of <t>HER2,</t> EGFR, and beta-actin in breast cancer cell lines, newly established cell lines, their corresponding PDX-derived organoids and PDX tumors. Expression of HER2 and EGFR for MC-BR-BTY-0019 cell line, organoid, and PDX was normalized to beta-actin and then SKBR3. Expression of HER2 and EGFR for MC-BR-BTY-0006 cell line, organoid, and PDX was normalized to beta-actin and then MDA-MB-231. All blots are derived from the same experiment and were processed in parallel. B MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with HER2 and DAPI and images were taken at ×100. Scale bar represents 10 μm. C MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with EGFR and DAPI and images were taken at ×100. Scale bar represents 10 μm. D 10,000 cells/well of MC-BR-BTY-0019 cell line were seeded in 96-well plates in triplicates, treated with indicated concentrations of lapatinib for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = 438.531 nM. E 10,000 cells/well of MC-BR-BTY-0006 cell line were seeded in 96-well plates in triplicates, treated with paclitaxel at indicated concentrations for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = Indeterminate. F , G PDX tumors were collected when the tumor achieved 10–20 mm in diameter, and primary breast cancer cells were isolated after dissociation of tumor tissue from mouse cells, as previously described . MC-BR-BTY-0019 PDX-derived organoids were generated by seeding 10,000 cells of the primary breast cancer single-cell suspension per well in Nanoculture plates in triplicates and treated with indicated concentrations of lapatinib for 5 days (IC 50 = 979.49 nM). MC-BR-BTY-0006 PDX-derived organoids were treated with indicated concentrations of paclitaxel for 5 days (IC 50 = Indeterminate). Organoid viability was measured using 3D Cell TiterGlo viability assay, normalized to DMSO, and plotted using GraphPad PRISM software. Error bars represent SEM.
    Her2 Erbb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Establishment and characterization of immortalized human breast cancer cell lines from breast cancer patient-derived xenografts (PDX)"

    Article Title: Establishment and characterization of immortalized human breast cancer cell lines from breast cancer patient-derived xenografts (PDX)

    Journal: NPJ Breast Cancer

    doi: 10.1038/s41523-021-00285-x

    A Western blot and quantification of the levels of HER2, EGFR, and beta-actin in breast cancer cell lines, newly established cell lines, their corresponding PDX-derived organoids and PDX tumors. Expression of HER2 and EGFR for MC-BR-BTY-0019 cell line, organoid, and PDX was normalized to beta-actin and then SKBR3. Expression of HER2 and EGFR for MC-BR-BTY-0006 cell line, organoid, and PDX was normalized to beta-actin and then MDA-MB-231. All blots are derived from the same experiment and were processed in parallel. B MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with HER2 and DAPI and images were taken at ×100. Scale bar represents 10 μm. C MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with EGFR and DAPI and images were taken at ×100. Scale bar represents 10 μm. D 10,000 cells/well of MC-BR-BTY-0019 cell line were seeded in 96-well plates in triplicates, treated with indicated concentrations of lapatinib for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = 438.531 nM. E 10,000 cells/well of MC-BR-BTY-0006 cell line were seeded in 96-well plates in triplicates, treated with paclitaxel at indicated concentrations for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = Indeterminate. F , G PDX tumors were collected when the tumor achieved 10–20 mm in diameter, and primary breast cancer cells were isolated after dissociation of tumor tissue from mouse cells, as previously described . MC-BR-BTY-0019 PDX-derived organoids were generated by seeding 10,000 cells of the primary breast cancer single-cell suspension per well in Nanoculture plates in triplicates and treated with indicated concentrations of lapatinib for 5 days (IC 50 = 979.49 nM). MC-BR-BTY-0006 PDX-derived organoids were treated with indicated concentrations of paclitaxel for 5 days (IC 50 = Indeterminate). Organoid viability was measured using 3D Cell TiterGlo viability assay, normalized to DMSO, and plotted using GraphPad PRISM software. Error bars represent SEM.
    Figure Legend Snippet: A Western blot and quantification of the levels of HER2, EGFR, and beta-actin in breast cancer cell lines, newly established cell lines, their corresponding PDX-derived organoids and PDX tumors. Expression of HER2 and EGFR for MC-BR-BTY-0019 cell line, organoid, and PDX was normalized to beta-actin and then SKBR3. Expression of HER2 and EGFR for MC-BR-BTY-0006 cell line, organoid, and PDX was normalized to beta-actin and then MDA-MB-231. All blots are derived from the same experiment and were processed in parallel. B MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with HER2 and DAPI and images were taken at ×100. Scale bar represents 10 μm. C MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with EGFR and DAPI and images were taken at ×100. Scale bar represents 10 μm. D 10,000 cells/well of MC-BR-BTY-0019 cell line were seeded in 96-well plates in triplicates, treated with indicated concentrations of lapatinib for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = 438.531 nM. E 10,000 cells/well of MC-BR-BTY-0006 cell line were seeded in 96-well plates in triplicates, treated with paclitaxel at indicated concentrations for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = Indeterminate. F , G PDX tumors were collected when the tumor achieved 10–20 mm in diameter, and primary breast cancer cells were isolated after dissociation of tumor tissue from mouse cells, as previously described . MC-BR-BTY-0019 PDX-derived organoids were generated by seeding 10,000 cells of the primary breast cancer single-cell suspension per well in Nanoculture plates in triplicates and treated with indicated concentrations of lapatinib for 5 days (IC 50 = 979.49 nM). MC-BR-BTY-0006 PDX-derived organoids were treated with indicated concentrations of paclitaxel for 5 days (IC 50 = Indeterminate). Organoid viability was measured using 3D Cell TiterGlo viability assay, normalized to DMSO, and plotted using GraphPad PRISM software. Error bars represent SEM.

    Techniques Used: Western Blot, Derivative Assay, Expressing, Staining, MTS Assay, Isolation, Generated, Viability Assay, Software

    A , B Pathology staining of HE, Ki67, ER, PR, EGFR, and HER2 was performed in both PDX tumor tissue and CDX tumor formed from MC-BR-BTY-0019 cell line ( A ) and MC-BR-BTY-0006 ( B ). Images taken at ×200; Scale bar represents 200 μm. C Detailed description of the pathology staining features of tumor tissue of the PDX and CDX is summarized for MC-BR-BTY-0019 and MC-BR-BTY-0006. D MC-BR-BTY-0019 CDX in vivo tumors were formed by injecting 2 million cells of MC-BR-BTY-0019 cell line into 10 NSG mice, which were randomized when tumor achieved 50–100 mm 3 in volume, and treated with either control ( n = 5, vehicle, 5 days/week) or lapatinib ( n = 5, 50 mg/kg, 5 days/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. ** p < 0.01, 2 tailed Student’s t -test comparing vehicle to drug treatment. E MC-BR-BTY-0019 PDX tumor was formed by injecting earlier passage of PDX tumor into flank of 15 NSG mice, 13 mice with similar size of tumors were randomized when tumor achieved 50–100 mm 3 in volume and treated with either control ( n = 6, vehicle, 5 days/week) or lapatinib ( n = 7, 50 mg/kg, 5 days/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. ** p < 0.01, 2 tailed Student’s t -test comparing vehicle to drug treatment. F MC-BR-BTY-0006 CDX in vivo tumors were formed by injecting 5 million cells of MC-BR-BTY-0006 cell line into 10 NSG mice, which were randomized when the tumor reached 50–100 mm 3 in volume, and treated with either control ( n = 5, vehicle, 2 times/week) or paclitaxel ( n = 5, 12.5 mg/kg, 2 times/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. p -value = ns, 2 tailed Student’s t -test comparing vehicle to drug treatment. G MC-BR-BTY-0006 PDX tumor was formed by injecting earlier passage of PDX tumor into flank of 15 NSG mice, 12 mice with similar size of tumor were randomized when tumor reached 50–100 mm 3 in volume, and treated with either control ( n = 6, vehicle, 2 times/week) or paclitaxel ( n = 6, 12.5 mg/kg, 2 times/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. p -value = ns, 2 tailed Student’s t -test comparing vehicle to drug treatment.
    Figure Legend Snippet: A , B Pathology staining of HE, Ki67, ER, PR, EGFR, and HER2 was performed in both PDX tumor tissue and CDX tumor formed from MC-BR-BTY-0019 cell line ( A ) and MC-BR-BTY-0006 ( B ). Images taken at ×200; Scale bar represents 200 μm. C Detailed description of the pathology staining features of tumor tissue of the PDX and CDX is summarized for MC-BR-BTY-0019 and MC-BR-BTY-0006. D MC-BR-BTY-0019 CDX in vivo tumors were formed by injecting 2 million cells of MC-BR-BTY-0019 cell line into 10 NSG mice, which were randomized when tumor achieved 50–100 mm 3 in volume, and treated with either control ( n = 5, vehicle, 5 days/week) or lapatinib ( n = 5, 50 mg/kg, 5 days/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. ** p < 0.01, 2 tailed Student’s t -test comparing vehicle to drug treatment. E MC-BR-BTY-0019 PDX tumor was formed by injecting earlier passage of PDX tumor into flank of 15 NSG mice, 13 mice with similar size of tumors were randomized when tumor achieved 50–100 mm 3 in volume and treated with either control ( n = 6, vehicle, 5 days/week) or lapatinib ( n = 7, 50 mg/kg, 5 days/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. ** p < 0.01, 2 tailed Student’s t -test comparing vehicle to drug treatment. F MC-BR-BTY-0006 CDX in vivo tumors were formed by injecting 5 million cells of MC-BR-BTY-0006 cell line into 10 NSG mice, which were randomized when the tumor reached 50–100 mm 3 in volume, and treated with either control ( n = 5, vehicle, 2 times/week) or paclitaxel ( n = 5, 12.5 mg/kg, 2 times/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. p -value = ns, 2 tailed Student’s t -test comparing vehicle to drug treatment. G MC-BR-BTY-0006 PDX tumor was formed by injecting earlier passage of PDX tumor into flank of 15 NSG mice, 12 mice with similar size of tumor were randomized when tumor reached 50–100 mm 3 in volume, and treated with either control ( n = 6, vehicle, 2 times/week) or paclitaxel ( n = 6, 12.5 mg/kg, 2 times/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. p -value = ns, 2 tailed Student’s t -test comparing vehicle to drug treatment.

    Techniques Used: Staining, In Vivo, Software

    A Oncogenic mutations in five cancer genes were detected in all three MC-BR-BTY-0006 tumor samples. Pathogenic loss-of-function somatic SNVs were detected in one allele of each of the genes TP53, PTEN, and BRCA2 as well as the other allele (loss of heterozygosity, or LOH). Thus, loss of function of both copies of tumor-suppressor genes TP53, PTEN, and BRCA2 could be inferred. RB1 loss of heterozygosity (LOH) and amplification of MYC were other oncogenic events observed. B Oncogenic mutations in eight cancer genes were detected in all three MC-BR-BTY-0019 tumor samples. A different pathogenic loss-of-function mutation in TP53 for cell line, but was again observed alongside LOH. So, loss of function of both copies can be inferred for TP53 as well as for three other tumor suppressor genes, CDKN2A, the adjacent CDKN2B, and MAP2K4, due to homozygous deletion (HomDel). Mutations were also observed in PIK3R1 and ARID1A. The mutations affect sites in regions frequently altered in cancer. Amplifications of TERT and ERBB2, or HER2, were also detected, with particularly high amplification for ERBB2. C Of the somatic SNV/INDELs detected in the patient tumor tissue samples, proportions detected in PDXs and cell lines were high (≥72%) for both patients. Of those mutations detected in PDXs, proportions detected in cell lines were also high (79%, 67%). D Changes in ploidy by sample type pair. For each patient, the absolute differences were nearly 1 copy from tissue to PDX with a Tissue→PDX increase observed for MC-BR-BTY-0006 and a Tissue→PDX decrease observed for MC-BR-BTY-0019. Despite these changes, the profiles of within-sample relative copy numbers for Tissue and PDX still showed similarity as the correlation of copy number was still 0.70 or greater. Slight decreases in ploidy were observed from PDX to cell line for both patients.
    Figure Legend Snippet: A Oncogenic mutations in five cancer genes were detected in all three MC-BR-BTY-0006 tumor samples. Pathogenic loss-of-function somatic SNVs were detected in one allele of each of the genes TP53, PTEN, and BRCA2 as well as the other allele (loss of heterozygosity, or LOH). Thus, loss of function of both copies of tumor-suppressor genes TP53, PTEN, and BRCA2 could be inferred. RB1 loss of heterozygosity (LOH) and amplification of MYC were other oncogenic events observed. B Oncogenic mutations in eight cancer genes were detected in all three MC-BR-BTY-0019 tumor samples. A different pathogenic loss-of-function mutation in TP53 for cell line, but was again observed alongside LOH. So, loss of function of both copies can be inferred for TP53 as well as for three other tumor suppressor genes, CDKN2A, the adjacent CDKN2B, and MAP2K4, due to homozygous deletion (HomDel). Mutations were also observed in PIK3R1 and ARID1A. The mutations affect sites in regions frequently altered in cancer. Amplifications of TERT and ERBB2, or HER2, were also detected, with particularly high amplification for ERBB2. C Of the somatic SNV/INDELs detected in the patient tumor tissue samples, proportions detected in PDXs and cell lines were high (≥72%) for both patients. Of those mutations detected in PDXs, proportions detected in cell lines were also high (79%, 67%). D Changes in ploidy by sample type pair. For each patient, the absolute differences were nearly 1 copy from tissue to PDX with a Tissue→PDX increase observed for MC-BR-BTY-0006 and a Tissue→PDX decrease observed for MC-BR-BTY-0019. Despite these changes, the profiles of within-sample relative copy numbers for Tissue and PDX still showed similarity as the correlation of copy number was still 0.70 or greater. Slight decreases in ploidy were observed from PDX to cell line for both patients.

    Techniques Used: Amplification, Mutagenesis

    anti her2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti her2
    Mutations in the PI3K pathway are associated with poor overall survival for patients with <t>HER2-positive</t> (HER2+) and HER2 and ER double-positive (HER2+/ER+) breast cancer. ( a ) Overall survival (OS) was analyzed in patients with HER2+ breast cancer with (red) or without (blue) mutations in the PI3K pathway. Median OS was 115.3 (blue) vs 79.5 months (red), respectively (hazard ratio: 1.82; 95% CI 1.0–3.3, p value = 0.036). ( b ) OS was analyzed in patients with HER2+/ER+ breast cancer with (red) or without (blue) mutations in PI3K pathway. Median OS was 115.3 (blue) vs 79.0 months (red), respectively (hazard ratio: 2.10; 95% CI: 1.0–4.5, p value = 0.04).
    Anti Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Combination treatment with a PI3K/Akt/mTOR pathway inhibitor overcomes resistance to anti-HER2 therapy in PIK3CA -mutant HER2-positive breast cancer cells"

    Article Title: Combination treatment with a PI3K/Akt/mTOR pathway inhibitor overcomes resistance to anti-HER2 therapy in PIK3CA -mutant HER2-positive breast cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78646-y

    Mutations in the PI3K pathway are associated with poor overall survival for patients with HER2-positive (HER2+) and HER2 and ER double-positive (HER2+/ER+) breast cancer. ( a ) Overall survival (OS) was analyzed in patients with HER2+ breast cancer with (red) or without (blue) mutations in the PI3K pathway. Median OS was 115.3 (blue) vs 79.5 months (red), respectively (hazard ratio: 1.82; 95% CI 1.0–3.3, p value = 0.036). ( b ) OS was analyzed in patients with HER2+/ER+ breast cancer with (red) or without (blue) mutations in PI3K pathway. Median OS was 115.3 (blue) vs 79.0 months (red), respectively (hazard ratio: 2.10; 95% CI: 1.0–4.5, p value = 0.04).
    Figure Legend Snippet: Mutations in the PI3K pathway are associated with poor overall survival for patients with HER2-positive (HER2+) and HER2 and ER double-positive (HER2+/ER+) breast cancer. ( a ) Overall survival (OS) was analyzed in patients with HER2+ breast cancer with (red) or without (blue) mutations in the PI3K pathway. Median OS was 115.3 (blue) vs 79.5 months (red), respectively (hazard ratio: 1.82; 95% CI 1.0–3.3, p value = 0.036). ( b ) OS was analyzed in patients with HER2+/ER+ breast cancer with (red) or without (blue) mutations in PI3K pathway. Median OS was 115.3 (blue) vs 79.0 months (red), respectively (hazard ratio: 2.10; 95% CI: 1.0–4.5, p value = 0.04).

    Techniques Used:

    The antiproliferative effect of treatment with fulvestrant and lapatinib is limited in MDA-MB-361 cell line. ( a ) Antiproliferative activity of fulvestrant and lapatinib, alone and in combination, in ER+/HER2+ breast cancer cell lines with and without PIK3CA mutations. BT474 (left) and MDA-MB-361 (right) cells were used as representative PIK3CA-wild-type and PIK3CA-mutant ER+/HER2 breast cancer cell lines, respectively. Cells were treated with DMSO (black), fulvestrant (100 nM, green), or lapatinib (100 nM, blue), alone and in combination (red), for 0, 2, 4, and 8 days (mean ± standard deviation [SD; n = 3]). Relative levels of ATP were calculated by chemiluminescence assay and compared with the chemiluminescence of DMSO on day 0. ( b ) Comparison of the effect in each drug treatment between BT474 and MDA-MB-361 cell lines. Differences on day 8 were analyzed using Student’s t-test. The data represent the mean ± SD (n = 3). F, Fulvestrant; L, Lapatinib.
    Figure Legend Snippet: The antiproliferative effect of treatment with fulvestrant and lapatinib is limited in MDA-MB-361 cell line. ( a ) Antiproliferative activity of fulvestrant and lapatinib, alone and in combination, in ER+/HER2+ breast cancer cell lines with and without PIK3CA mutations. BT474 (left) and MDA-MB-361 (right) cells were used as representative PIK3CA-wild-type and PIK3CA-mutant ER+/HER2 breast cancer cell lines, respectively. Cells were treated with DMSO (black), fulvestrant (100 nM, green), or lapatinib (100 nM, blue), alone and in combination (red), for 0, 2, 4, and 8 days (mean ± standard deviation [SD; n = 3]). Relative levels of ATP were calculated by chemiluminescence assay and compared with the chemiluminescence of DMSO on day 0. ( b ) Comparison of the effect in each drug treatment between BT474 and MDA-MB-361 cell lines. Differences on day 8 were analyzed using Student’s t-test. The data represent the mean ± SD (n = 3). F, Fulvestrant; L, Lapatinib.

    Techniques Used: Activity Assay, Mutagenesis, Standard Deviation, Chemiluminescence Immunoassay

    Lapatinib suppresses 4EBP1 phosphorylation insufficiently in MDA-MB-361 cells and the antiproliferative effect of lapatinib is limited in HER2+ breast cancer cell lines with PIK3CA mutations. ( a ) Inhibitory effects of fulvestrant (100 nM) and lapatinib (100 nM), alone and in combination treated for 24 h, on 4EBP1 phosphorylation in ER+/HER2+ breast cancer cell lines with and without PIK3CA mutation. Full-length blots are presented in Supplementary <xref ref-type=Fig. 3 . ( b ) Newly synthesized protein in DMSO-, fulvestrant-, lapatinib-, and combination-treated cells. BT474 (upper) and MDA-MB-361 (lower) cells were treated with the indicated inhibitors for 24 h; Click-iT HPG was incorporated into the drug-treated cells for 15 h. Green indicates the newly synthesized proteins detected by the Click-iT assay (Life Technologies) and DAPI-stained nuclei, respectively. ( c ) PIK3CA mutation, ER expression, HER2 amplification, PTEN Loss and PIK3R1 status of the breast cancer cell lines used in this study. Information on gene modification and expression was obtained from the COSMIC database and ATCC information. ( d ) Antiproliferative activity of combined fulvestrant and lapatinib in HER2+ breast cancer cell lines with and without PIK3CA mutation. Three PIK3CA-wild-type (BT474, SK-BR-3, and ZR-75-30; red bars) and three PIK3CA-mutant (UACC893, HCC1954, and MDA-MB-361; blue bars) HER2+ breast cancer cell lines were used. The cells were treated with fulvestrant and lapatinib in combination for 8 days (mean ± SD [n = 3]). Y-axis indicates relative amounts of ATP (%), which were calculated with a chemiluminescence assay and compared with the chemiluminescence value of DMSO treatment on day 8. The data represent the mean ± SD (n = 3). ( e ) Inhibitory effects of fulvestrant (100 nM) and lapatinib (100 nM) in combination treatment for 24 h, on 4EBP1 phosphorylation in HER2+ breast cancer cell lines with and without PIK3CA mutation. Three PIK3CA-wild-type (BT474, SK-BR-3, and ZR-75-30) (left panel) and three PIK3CA-mutant (UACC893, HCC1954, and MDA-MB-361) (right panel) HER2+ breast cancer cell lines were used. Full-length blots are presented in Supplementary Fig. . " title="... the antiproliferative effect of lapatinib is limited in HER2+ breast cancer cell lines with PIK3CA mutations. ( ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Lapatinib suppresses 4EBP1 phosphorylation insufficiently in MDA-MB-361 cells and the antiproliferative effect of lapatinib is limited in HER2+ breast cancer cell lines with PIK3CA mutations. ( a ) Inhibitory effects of fulvestrant (100 nM) and lapatinib (100 nM), alone and in combination treated for 24 h, on 4EBP1 phosphorylation in ER+/HER2+ breast cancer cell lines with and without PIK3CA mutation. Full-length blots are presented in Supplementary Fig. 3 . ( b ) Newly synthesized protein in DMSO-, fulvestrant-, lapatinib-, and combination-treated cells. BT474 (upper) and MDA-MB-361 (lower) cells were treated with the indicated inhibitors for 24 h; Click-iT HPG was incorporated into the drug-treated cells for 15 h. Green indicates the newly synthesized proteins detected by the Click-iT assay (Life Technologies) and DAPI-stained nuclei, respectively. ( c ) PIK3CA mutation, ER expression, HER2 amplification, PTEN Loss and PIK3R1 status of the breast cancer cell lines used in this study. Information on gene modification and expression was obtained from the COSMIC database and ATCC information. ( d ) Antiproliferative activity of combined fulvestrant and lapatinib in HER2+ breast cancer cell lines with and without PIK3CA mutation. Three PIK3CA-wild-type (BT474, SK-BR-3, and ZR-75-30; red bars) and three PIK3CA-mutant (UACC893, HCC1954, and MDA-MB-361; blue bars) HER2+ breast cancer cell lines were used. The cells were treated with fulvestrant and lapatinib in combination for 8 days (mean ± SD [n = 3]). Y-axis indicates relative amounts of ATP (%), which were calculated with a chemiluminescence assay and compared with the chemiluminescence value of DMSO treatment on day 8. The data represent the mean ± SD (n = 3). ( e ) Inhibitory effects of fulvestrant (100 nM) and lapatinib (100 nM) in combination treatment for 24 h, on 4EBP1 phosphorylation in HER2+ breast cancer cell lines with and without PIK3CA mutation. Three PIK3CA-wild-type (BT474, SK-BR-3, and ZR-75-30) (left panel) and three PIK3CA-mutant (UACC893, HCC1954, and MDA-MB-361) (right panel) HER2+ breast cancer cell lines were used. Full-length blots are presented in Supplementary Fig. .

    Techniques Used: Mutagenesis, Synthesized, Staining, Expressing, Amplification, Modification, Activity Assay, Chemiluminescence Immunoassay

    Ipatasertib enhances the antiproliferative activity of fulvestrant and lapatinib combination in a PIK3CA-mutant HER2+/ER+ breast cancer cell line. ( a ) Antiproliferative activity of BT474 (PIK3CA-wild-type, left) and MDA-MB-361 (PIK3CA-mutant, right) cells treated with DMSO (black), combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) (red) for 0, 2, 4, and 8 days (mean ± SD [n = 3]). Relative ATP amounts were compared with the chemiluminescence value of DMSO treatment on day 0. ( b ) Comparison of the inhibitory effect in each drug treatment between BT474 and MDA-MB-361 cell lines. Differences on day8 were analyzed using Student’s t-test. The data represent the mean ± SD (n = 3). ( c ) Representative images of crystal violet staining in BT474 or MDA-MB-361 cells treated with DMSO, the combination of fulvestrant (100 nM) and lapatinib (100 nM), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) for 8 days. ( d ) Quantified data from Fig. 4c. Crystal violet absorbance indicating the amount of normalized protein was measured with a microplate reader and compared with a DMSO control. The data represent mean ± SD (n = 3). Crystal violet absorbance was statistically analyzed using Student’s t-test to compare the double-combination and the triple-combination treatments. Differences were considered significant at p ≤ 0.05 (*). ( e ) Effect of combined fulvestrant, lapatinib, and ipatasertib treatment on the apoptosis of BT474 (left) and MDA-MB-361 (right) cells. The cells were treated with DMSO (black), the combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) (red) for 24 h (mean ± SD [n = 3]). Relative caspase-3/7 activities were calculated based on luminescence compared with the DMSO control. Caspase-3/7 activities were analyzed using Student’s t-test to compare the DMSO treatment and the double-combination treatment, and the double-combination and the triple-combination treatments. Differences were considered significant at p ≤ 0.05 (*). F, Fulvestrant; L, Lapatinib; I, Ipatasertib; n.s., not significant. ( f ) Representative images of 3D culture in BT474 (left) or MDA-MB-361 (right) cells. ( g ) Antiproliferative activity of 3D-cultured BT474 (PIK3CA-wild-type, left) and MDA-MB-361 (PIK3CA-mutant, right) cells treated with DMSO (black), combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) (red) for 0, 2, 4, and 8 days (mean ± SD [n = 3]). Relative ATP amounts were compared with the chemiluminescence value of DMSO treatment on day 0.
    Figure Legend Snippet: Ipatasertib enhances the antiproliferative activity of fulvestrant and lapatinib combination in a PIK3CA-mutant HER2+/ER+ breast cancer cell line. ( a ) Antiproliferative activity of BT474 (PIK3CA-wild-type, left) and MDA-MB-361 (PIK3CA-mutant, right) cells treated with DMSO (black), combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) (red) for 0, 2, 4, and 8 days (mean ± SD [n = 3]). Relative ATP amounts were compared with the chemiluminescence value of DMSO treatment on day 0. ( b ) Comparison of the inhibitory effect in each drug treatment between BT474 and MDA-MB-361 cell lines. Differences on day8 were analyzed using Student’s t-test. The data represent the mean ± SD (n = 3). ( c ) Representative images of crystal violet staining in BT474 or MDA-MB-361 cells treated with DMSO, the combination of fulvestrant (100 nM) and lapatinib (100 nM), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) for 8 days. ( d ) Quantified data from Fig. 4c. Crystal violet absorbance indicating the amount of normalized protein was measured with a microplate reader and compared with a DMSO control. The data represent mean ± SD (n = 3). Crystal violet absorbance was statistically analyzed using Student’s t-test to compare the double-combination and the triple-combination treatments. Differences were considered significant at p ≤ 0.05 (*). ( e ) Effect of combined fulvestrant, lapatinib, and ipatasertib treatment on the apoptosis of BT474 (left) and MDA-MB-361 (right) cells. The cells were treated with DMSO (black), the combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) (red) for 24 h (mean ± SD [n = 3]). Relative caspase-3/7 activities were calculated based on luminescence compared with the DMSO control. Caspase-3/7 activities were analyzed using Student’s t-test to compare the DMSO treatment and the double-combination treatment, and the double-combination and the triple-combination treatments. Differences were considered significant at p ≤ 0.05 (*). F, Fulvestrant; L, Lapatinib; I, Ipatasertib; n.s., not significant. ( f ) Representative images of 3D culture in BT474 (left) or MDA-MB-361 (right) cells. ( g ) Antiproliferative activity of 3D-cultured BT474 (PIK3CA-wild-type, left) and MDA-MB-361 (PIK3CA-mutant, right) cells treated with DMSO (black), combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) (red) for 0, 2, 4, and 8 days (mean ± SD [n = 3]). Relative ATP amounts were compared with the chemiluminescence value of DMSO treatment on day 0.

    Techniques Used: Activity Assay, Mutagenesis, Staining, Cell Culture

    Ipatasertib potently suppresses 4EBP1 phosphorylation in MDA-MB-361 cells treated with combined fulvestrant and lapatinib, inhibiting newly-synthesized proteins. ( a ) Inhibitory effects of ipatasertib on 4EBP1 phosphorylation in fulvestrant and lapatinib-treated BT474 (left) and MDA-MB-361 (right) cells. The cells were treated with fulvestrant (100 nM), lapatinib (100 nM), ipatasertib (1000 nM), and their combinations for 24 h. Plus (+) and minus (−) indicate the presence or absence of treatment. Phosphorylated HER2, ER, and AKT were used as pharmacodynamic markers to confirm target engagement of lapatinib, fulvestrant, and ipatasertib, respectively. β-actin was used as a loading control. Full-length blots are presented in Supplementary <xref ref-type=Figs. 8 and . ( b ) Newly synthesized protein in cells treated with DMSO, combination treatment with fulvestrant and lapatinib, and triple-combination treatment with fulvestrant, lapatinib, and ipatasertib. BT474 (left) and MDA-MB-361 (right) cells were treated with the indicated inhibitors for 24 h, and then Click-iT HPG was incorporated into drug-treated cells for 15 h. Green indicates the newly synthesized proteins detected by the Click-iT assay (Life Technologies) and DAPI-stained nuclei, respectively. ( c ) Quantified data from Fig. 5b. The fluorescent images were captured by BZ-X800 (Keyence Corporation), and the captured images were quantified by a BZ-X800 Analyzer (Keyence Corporation). Alexa-488 fluorescence was normalized using DAPI fluorescence. The y axis indicates the relative Alexa-488 fluorescence, calculated based on fluorescence compared with the DMSO-treatment control. The data represent the mean ± SD (n = 3). The relative Alexa-488 fluorescence was statistically analyzed using Student’s t-test to compare the double-combination and the triple-combination. Differences were considered significant at p ≤ 0.05 (*). F, Fulvestrant; L, Lapatinib; I, Ipatasertib; n.s., not significant. ( d ) Experimental schemes of cap-pulldown assay. ( e ) Cap pulldown assay in MDA-MB-361 cells in the triple combination. The cells were treated with fulvestrant (100 nM), lapatinib (100 nM), ipatasertib (1000 nM), and their combinations for 24 h. Immunoblotting of eIF4G, eIF4A, eIF4E, and 4EBP1 were performed in the m7 GTP pulldown samples. eIF4E was used as a pulldown positive control. Immunoblotting of eIF4G, eIF4A, eIF4E, p4EBP1, and 4EBP1 in cell lysates was performed as input controls. β-actin was used as a loading control. Full-length blots are presented in Supplementary Fig. 10 . " title="... indicate the presence or absence of treatment. Phosphorylated HER2, ER, and AKT were used as pharmacodynamic markers ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Ipatasertib potently suppresses 4EBP1 phosphorylation in MDA-MB-361 cells treated with combined fulvestrant and lapatinib, inhibiting newly-synthesized proteins. ( a ) Inhibitory effects of ipatasertib on 4EBP1 phosphorylation in fulvestrant and lapatinib-treated BT474 (left) and MDA-MB-361 (right) cells. The cells were treated with fulvestrant (100 nM), lapatinib (100 nM), ipatasertib (1000 nM), and their combinations for 24 h. Plus (+) and minus (−) indicate the presence or absence of treatment. Phosphorylated HER2, ER, and AKT were used as pharmacodynamic markers to confirm target engagement of lapatinib, fulvestrant, and ipatasertib, respectively. β-actin was used as a loading control. Full-length blots are presented in Supplementary Figs. 8 and . ( b ) Newly synthesized protein in cells treated with DMSO, combination treatment with fulvestrant and lapatinib, and triple-combination treatment with fulvestrant, lapatinib, and ipatasertib. BT474 (left) and MDA-MB-361 (right) cells were treated with the indicated inhibitors for 24 h, and then Click-iT HPG was incorporated into drug-treated cells for 15 h. Green indicates the newly synthesized proteins detected by the Click-iT assay (Life Technologies) and DAPI-stained nuclei, respectively. ( c ) Quantified data from Fig. 5b. The fluorescent images were captured by BZ-X800 (Keyence Corporation), and the captured images were quantified by a BZ-X800 Analyzer (Keyence Corporation). Alexa-488 fluorescence was normalized using DAPI fluorescence. The y axis indicates the relative Alexa-488 fluorescence, calculated based on fluorescence compared with the DMSO-treatment control. The data represent the mean ± SD (n = 3). The relative Alexa-488 fluorescence was statistically analyzed using Student’s t-test to compare the double-combination and the triple-combination. Differences were considered significant at p ≤ 0.05 (*). F, Fulvestrant; L, Lapatinib; I, Ipatasertib; n.s., not significant. ( d ) Experimental schemes of cap-pulldown assay. ( e ) Cap pulldown assay in MDA-MB-361 cells in the triple combination. The cells were treated with fulvestrant (100 nM), lapatinib (100 nM), ipatasertib (1000 nM), and their combinations for 24 h. Immunoblotting of eIF4G, eIF4A, eIF4E, and 4EBP1 were performed in the m7 GTP pulldown samples. eIF4E was used as a pulldown positive control. Immunoblotting of eIF4G, eIF4A, eIF4E, p4EBP1, and 4EBP1 in cell lysates was performed as input controls. β-actin was used as a loading control. Full-length blots are presented in Supplementary Fig. 10 .

    Techniques Used: Synthesized, Staining, Fluorescence, Western Blot, Positive Control

    Combination treatment with lapatinib and ipatasertib enhances antiproliferative activity in PIK3CA -mutant HER2+ breast cancer cell lines. ( a ) Antiproliferative activity of combination treatment with fulvestrant and lapatinib, and triple-combination treatment with fulvestrant, lapatinib, and ipatasertib in PIK3CA -wild-type and PIK3CA -mutant HER2+ breast cancer cell lines. Three PIK3CA-wild-type (BT474, SK-BR-3, and ZR-75-30; left) and three PIK3CA-mutant (UACC893, HCC1954, and MDA-MB-361; right) HER2+ breast cancer cell lines were used. Cells were treated with fulvestrant and lapatinib (blue), or triple-combination treatment with fulvestrant, lapatinib, and ipatasertib (red) for 8 days (mean ± SD [n = 3]). The Y-axis indicates the relative ATP amounts (%). The relative ATP amounts were calculated using chemiluminescence assay and compared with the chemiluminescence of the DMSO control on day 8. ( b ) Quantified data of ipatasertib combination effects form Fig. 6a. The effects of ipatasertib were calculated using the following formula: ipatasertib combination effects = (%ATP with triple-combination treatment with fulvestrant, lapatinib, and ipatasertib)—(%ATP with fulvestrant and lapatinib treatment). Statistical analyses were performed using the non-parametric Wilcoxon–Mann–Whitney test. Differences were considered significant at p < 0.05. F, Fulvestrant; L, Lapatinib; I, Ipatasertib.
    Figure Legend Snippet: Combination treatment with lapatinib and ipatasertib enhances antiproliferative activity in PIK3CA -mutant HER2+ breast cancer cell lines. ( a ) Antiproliferative activity of combination treatment with fulvestrant and lapatinib, and triple-combination treatment with fulvestrant, lapatinib, and ipatasertib in PIK3CA -wild-type and PIK3CA -mutant HER2+ breast cancer cell lines. Three PIK3CA-wild-type (BT474, SK-BR-3, and ZR-75-30; left) and three PIK3CA-mutant (UACC893, HCC1954, and MDA-MB-361; right) HER2+ breast cancer cell lines were used. Cells were treated with fulvestrant and lapatinib (blue), or triple-combination treatment with fulvestrant, lapatinib, and ipatasertib (red) for 8 days (mean ± SD [n = 3]). The Y-axis indicates the relative ATP amounts (%). The relative ATP amounts were calculated using chemiluminescence assay and compared with the chemiluminescence of the DMSO control on day 8. ( b ) Quantified data of ipatasertib combination effects form Fig. 6a. The effects of ipatasertib were calculated using the following formula: ipatasertib combination effects = (%ATP with triple-combination treatment with fulvestrant, lapatinib, and ipatasertib)—(%ATP with fulvestrant and lapatinib treatment). Statistical analyses were performed using the non-parametric Wilcoxon–Mann–Whitney test. Differences were considered significant at p < 0.05. F, Fulvestrant; L, Lapatinib; I, Ipatasertib.

    Techniques Used: Activity Assay, Mutagenesis, Chemiluminescence Immunoassay, MANN-WHITNEY

    rapamycin mtor  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rapamycin mtor
    (A and B) ARPE-19 cells were treated with 0.1 mM or 0.5 mM H 2 O 2 for the indicated times. (A) Cell death was analyzed by flow cytometry using propidium iodide staining. (B) The cell lysates were immunoblotted with the indicated antibodies. (C) ARPE-19 cells were treated with 0.5 mM H 2 O 2 in the presence or absence of the autophagy inhibitors 3-methyladenine (3-MA, 10 µM) and bafilomycin A1 (BafA1, 100 nM). The cell lysates were immunoblotted with the indicated antibodies. Cont, control. (B and C) LC3-II/-I ratio was quantified by densitometric analyses. (D) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 or autophagy modulators (inducer: <t>rapamycin,</t> 100 nM, or inhibitor: BafA1, 100 nM) for 6 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Scale bars = 10 µm. (A and D) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t-test. *P < 0.05, **P < 0.01.
    Rapamycin Mtor, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "PARP1 Impedes SIRT1-Mediated Autophagy during Degeneration of the Retinal Pigment Epithelium under Oxidative Stress"

    Article Title: PARP1 Impedes SIRT1-Mediated Autophagy during Degeneration of the Retinal Pigment Epithelium under Oxidative Stress

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2020.0078

    (A and B) ARPE-19 cells were treated with 0.1 mM or 0.5 mM H 2 O 2 for the indicated times. (A) Cell death was analyzed by flow cytometry using propidium iodide staining. (B) The cell lysates were immunoblotted with the indicated antibodies. (C) ARPE-19 cells were treated with 0.5 mM H 2 O 2 in the presence or absence of the autophagy inhibitors 3-methyladenine (3-MA, 10 µM) and bafilomycin A1 (BafA1, 100 nM). The cell lysates were immunoblotted with the indicated antibodies. Cont, control. (B and C) LC3-II/-I ratio was quantified by densitometric analyses. (D) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 or autophagy modulators (inducer: rapamycin, 100 nM, or inhibitor: BafA1, 100 nM) for 6 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Scale bars = 10 µm. (A and D) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t-test. *P < 0.05, **P < 0.01.
    Figure Legend Snippet: (A and B) ARPE-19 cells were treated with 0.1 mM or 0.5 mM H 2 O 2 for the indicated times. (A) Cell death was analyzed by flow cytometry using propidium iodide staining. (B) The cell lysates were immunoblotted with the indicated antibodies. (C) ARPE-19 cells were treated with 0.5 mM H 2 O 2 in the presence or absence of the autophagy inhibitors 3-methyladenine (3-MA, 10 µM) and bafilomycin A1 (BafA1, 100 nM). The cell lysates were immunoblotted with the indicated antibodies. Cont, control. (B and C) LC3-II/-I ratio was quantified by densitometric analyses. (D) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 or autophagy modulators (inducer: rapamycin, 100 nM, or inhibitor: BafA1, 100 nM) for 6 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Scale bars = 10 µm. (A and D) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t-test. *P < 0.05, **P < 0.01.

    Techniques Used: Flow Cytometry, Staining, Transfection

    (A) ARPE-19 cells were treated with 0.5 mM H 2 O 2 for the indicated times in the presence or absence of 10 µM olaparib (Ola). Veh, vehicle. (B and C) ARPE-19 cells were transfected with scrambled siRNA (scRNA) or ATG7 targeting siRNA for 48 h. Then, the cells were treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (B) for indicated times (C). (D and F) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (D) or pretreated with 100 µM rapamycin and then treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (F). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Cont, control. Scale bars = 10 µm. (E and F) ARPE-19 cells were pretreated with 100 µM rapamycin for 5 h and then treated with 0.5 mM H 2 O 2 for 1 h in the presence or absence of 10 µM olaparib. (A, B, C, and E) The cell lysates were immunoblotted with the indicated antibodies. The LC3-II/-I ratio and relative p62 levels were quantified by densitometric analyses (ImageJ software). (D and F) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001.
    Figure Legend Snippet: (A) ARPE-19 cells were treated with 0.5 mM H 2 O 2 for the indicated times in the presence or absence of 10 µM olaparib (Ola). Veh, vehicle. (B and C) ARPE-19 cells were transfected with scrambled siRNA (scRNA) or ATG7 targeting siRNA for 48 h. Then, the cells were treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (B) for indicated times (C). (D and F) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (D) or pretreated with 100 µM rapamycin and then treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (F). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Cont, control. Scale bars = 10 µm. (E and F) ARPE-19 cells were pretreated with 100 µM rapamycin for 5 h and then treated with 0.5 mM H 2 O 2 for 1 h in the presence or absence of 10 µM olaparib. (A, B, C, and E) The cell lysates were immunoblotted with the indicated antibodies. The LC3-II/-I ratio and relative p62 levels were quantified by densitometric analyses (ImageJ software). (D and F) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001.

    Techniques Used: Transfection, Staining, Software

    anti her2 44e7  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti her2 44e7
    Anti Her2 44e7, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antiphospho ser 2248 mtor  (Cell Signaling Technology Inc)


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    Antiphospho Ser 2248 Mtor, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    her2  (Cell Signaling Technology Inc)


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    Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse erbb2
    A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to <t>ERBB2</t> was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.
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    Zotatifin translationally downregulates FGFR1/2 and <t>HER2</t> in RTK-driven tumors. (A) Schematic of RTK-stimulated PI3K/AKT and RAS/ERK pathways that activate eIF4A. (B) Frequency of HER2 and FGFR1/2 dysregulation in different cancer types. (C) Western blot analysis of RTKs and cyclin D1 levels following 24 h treatment with zotatifin or DMSO in HER2- or FGFR1/2-driven tumor models. β-actin or α-tubulin serve as loading controls. (D) Polysome profiling and mRNA distribution in MDA-MB-361 breast cancer cells treated for 3 h with 20 nM zotatifin compared to DMSO. mRNA levels of HER2 are monitored in polysome fractions. GAPDH serves as a control. (E) Luciferase reporter assay with gene constructs containing 5’-UTRs of FGFR1/2, HER2 or TUBA in HEK293 cells following 4 h of dose-dependent treatment with zotatifin. (F) Left, list of genes of which their 5’-UTR sequences were cloned into luciferase reporter constructs. Right, measured IC 50 values (nM) for inhibition of expression following 4 h treatment with zotatifin.
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    Cell Signaling Technology Inc anti her2
    TRIB3 and <t>HER2</t> expression exhibit a positive correlation in breast cancer. ( a ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in a panel of breast cancer cell lines (RNA seq data were obtained from the GSE48216 dataset). ( n = 56; p and R were calculated using Spearman’s correlation coefficient). ( b ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in breast cancer patients (RNA seq data were obtained from the TGCA Breast cancer dataset using the UCSC Xena platform) ( n = 1247; p and R were calculated using the Spearman correlation coefficient). ( c ) Correlation between TRIB3 protein levels (as determined by TRIB3 immunostaining and the subsequent assignment of the corresponding H value) with respect to HER2 levels (pathologically classified as 0-1-2-3 according to the level of expression of this receptor) in a tissue microarray that was generated with samples that were derived from luminal breast cancer patients ( n = 159; p was calculated using the Spearman’s correlation coefficient).
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    Cell Signaling Technology Inc her2
    A BT474 cells were treated with Leptin and AG1478 alone or in combination for 24 h. Cell lysates were immunoblotted with indicated antibodies. B BT474 cells were treated with Leptin and AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMed1, pHER2, and tHER2. Actin was used as loading control. C Bar graph shows fold change in protein expression in ( A ) and ( B ). D Immunoblot analysis of MCF7-vector and <t>MCF7-Her2</t> O/E cells for indicated proteins. E , F MCF7-HER2 O/E cells were treated with Leptin, AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMED1, pHER2, and tHER2. Actin was used as loading control. Bar graph shows the ratio of MED1 and actin. G MCF7 and BT474 cells were treated as in ( A ) and ( B ), and subjected to immunofluorescence analysis of pMED1. Scale bar, 10 µm. H MCF7 cells were treated as indicated for 24 h and subjected to ChIP assay using Med1 antibody. The purified DNA was analyzed by using specific primer for ER promoter. ** p < 0.005, * p < 0.05. Data are means ± SD from three experiments.
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    Cell Signaling Technology Inc her2 erbb2
    A Western blot and quantification of the levels of <t>HER2,</t> EGFR, and beta-actin in breast cancer cell lines, newly established cell lines, their corresponding PDX-derived organoids and PDX tumors. Expression of HER2 and EGFR for MC-BR-BTY-0019 cell line, organoid, and PDX was normalized to beta-actin and then SKBR3. Expression of HER2 and EGFR for MC-BR-BTY-0006 cell line, organoid, and PDX was normalized to beta-actin and then MDA-MB-231. All blots are derived from the same experiment and were processed in parallel. B MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with HER2 and DAPI and images were taken at ×100. Scale bar represents 10 μm. C MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with EGFR and DAPI and images were taken at ×100. Scale bar represents 10 μm. D 10,000 cells/well of MC-BR-BTY-0019 cell line were seeded in 96-well plates in triplicates, treated with indicated concentrations of lapatinib for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = 438.531 nM. E 10,000 cells/well of MC-BR-BTY-0006 cell line were seeded in 96-well plates in triplicates, treated with paclitaxel at indicated concentrations for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = Indeterminate. F , G PDX tumors were collected when the tumor achieved 10–20 mm in diameter, and primary breast cancer cells were isolated after dissociation of tumor tissue from mouse cells, as previously described . MC-BR-BTY-0019 PDX-derived organoids were generated by seeding 10,000 cells of the primary breast cancer single-cell suspension per well in Nanoculture plates in triplicates and treated with indicated concentrations of lapatinib for 5 days (IC 50 = 979.49 nM). MC-BR-BTY-0006 PDX-derived organoids were treated with indicated concentrations of paclitaxel for 5 days (IC 50 = Indeterminate). Organoid viability was measured using 3D Cell TiterGlo viability assay, normalized to DMSO, and plotted using GraphPad PRISM software. Error bars represent SEM.
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    Cell Signaling Technology Inc rapamycin mtor
    (A and B) ARPE-19 cells were treated with 0.1 mM or 0.5 mM H 2 O 2 for the indicated times. (A) Cell death was analyzed by flow cytometry using propidium iodide staining. (B) The cell lysates were immunoblotted with the indicated antibodies. (C) ARPE-19 cells were treated with 0.5 mM H 2 O 2 in the presence or absence of the autophagy inhibitors 3-methyladenine (3-MA, 10 µM) and bafilomycin A1 (BafA1, 100 nM). The cell lysates were immunoblotted with the indicated antibodies. Cont, control. (B and C) LC3-II/-I ratio was quantified by densitometric analyses. (D) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 or autophagy modulators (inducer: <t>rapamycin,</t> 100 nM, or inhibitor: BafA1, 100 nM) for 6 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Scale bars = 10 µm. (A and D) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t-test. *P < 0.05, **P < 0.01.
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    Cell Signaling Technology Inc anti her2 44e7
    (A and B) ARPE-19 cells were treated with 0.1 mM or 0.5 mM H 2 O 2 for the indicated times. (A) Cell death was analyzed by flow cytometry using propidium iodide staining. (B) The cell lysates were immunoblotted with the indicated antibodies. (C) ARPE-19 cells were treated with 0.5 mM H 2 O 2 in the presence or absence of the autophagy inhibitors 3-methyladenine (3-MA, 10 µM) and bafilomycin A1 (BafA1, 100 nM). The cell lysates were immunoblotted with the indicated antibodies. Cont, control. (B and C) LC3-II/-I ratio was quantified by densitometric analyses. (D) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 or autophagy modulators (inducer: <t>rapamycin,</t> 100 nM, or inhibitor: BafA1, 100 nM) for 6 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Scale bars = 10 µm. (A and D) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t-test. *P < 0.05, **P < 0.01.
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    Cell Signaling Technology Inc antiphospho ser 2248 mtor
    (A and B) ARPE-19 cells were treated with 0.1 mM or 0.5 mM H 2 O 2 for the indicated times. (A) Cell death was analyzed by flow cytometry using propidium iodide staining. (B) The cell lysates were immunoblotted with the indicated antibodies. (C) ARPE-19 cells were treated with 0.5 mM H 2 O 2 in the presence or absence of the autophagy inhibitors 3-methyladenine (3-MA, 10 µM) and bafilomycin A1 (BafA1, 100 nM). The cell lysates were immunoblotted with the indicated antibodies. Cont, control. (B and C) LC3-II/-I ratio was quantified by densitometric analyses. (D) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 or autophagy modulators (inducer: <t>rapamycin,</t> 100 nM, or inhibitor: BafA1, 100 nM) for 6 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Scale bars = 10 µm. (A and D) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t-test. *P < 0.05, **P < 0.01.
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    Image Search Results


    A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to ERBB2 was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.

    Journal: PLoS ONE

    Article Title: Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies

    doi: 10.1371/journal.pone.0112376

    Figure Lengend Snippet: A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to ERBB2 was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.

    Article Snippet: Rabbit phospho-ERBB3 (Y1289) (#4791), ERBB3 (#12708P), phospho-ERBB2 (Y1221/1222) (#2243), phospho-Akt (Ser473) (#4060) and mouse ERBB2 (#2248) and Akt (pan) (#2920) antibodies were from Cell Signaling Technology.

    Techniques: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Standard Deviation, Incubation, Flow Cytometry, Positive Control, Negative Control

    Individual, pairwise, and triple combinations of A5, F4 and either trastuzumab or erlotinib (as appropriate) were serially diluted (2-fold dilution scheme) and incubated with the ERBB2-positive cell lines, BT-474, SK-BR-3, NCI-N87, and EGFR-positive cell line ACHN, as noted. Concentration plotted on the x-axis refers to anti-ERBB3 IgG concentration. Trastuzumab and erlotinib are at 1∶200 molar ratios with anti-ERBB3 antibodies. The impact of treatment on cell viability was quantified with Cell-Titer Blue. Vehicle-treated controls for each cell line were set at 100%. Values plotted represent the means ± standard error of the means (SEM) for between 2–6 independent experiments, each carried out in triplicate.

    Journal: PLoS ONE

    Article Title: Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies

    doi: 10.1371/journal.pone.0112376

    Figure Lengend Snippet: Individual, pairwise, and triple combinations of A5, F4 and either trastuzumab or erlotinib (as appropriate) were serially diluted (2-fold dilution scheme) and incubated with the ERBB2-positive cell lines, BT-474, SK-BR-3, NCI-N87, and EGFR-positive cell line ACHN, as noted. Concentration plotted on the x-axis refers to anti-ERBB3 IgG concentration. Trastuzumab and erlotinib are at 1∶200 molar ratios with anti-ERBB3 antibodies. The impact of treatment on cell viability was quantified with Cell-Titer Blue. Vehicle-treated controls for each cell line were set at 100%. Values plotted represent the means ± standard error of the means (SEM) for between 2–6 independent experiments, each carried out in triplicate.

    Article Snippet: Rabbit phospho-ERBB3 (Y1289) (#4791), ERBB3 (#12708P), phospho-ERBB2 (Y1221/1222) (#2243), phospho-Akt (Ser473) (#4060) and mouse ERBB2 (#2248) and Akt (pan) (#2920) antibodies were from Cell Signaling Technology.

    Techniques: Incubation, Concentration Assay

    Zotatifin translationally downregulates FGFR1/2 and HER2 in RTK-driven tumors. (A) Schematic of RTK-stimulated PI3K/AKT and RAS/ERK pathways that activate eIF4A. (B) Frequency of HER2 and FGFR1/2 dysregulation in different cancer types. (C) Western blot analysis of RTKs and cyclin D1 levels following 24 h treatment with zotatifin or DMSO in HER2- or FGFR1/2-driven tumor models. β-actin or α-tubulin serve as loading controls. (D) Polysome profiling and mRNA distribution in MDA-MB-361 breast cancer cells treated for 3 h with 20 nM zotatifin compared to DMSO. mRNA levels of HER2 are monitored in polysome fractions. GAPDH serves as a control. (E) Luciferase reporter assay with gene constructs containing 5’-UTRs of FGFR1/2, HER2 or TUBA in HEK293 cells following 4 h of dose-dependent treatment with zotatifin. (F) Left, list of genes of which their 5’-UTR sequences were cloned into luciferase reporter constructs. Right, measured IC 50 values (nM) for inhibition of expression following 4 h treatment with zotatifin.

    Journal: Frontiers in Oncology

    Article Title: Zotatifin, an eIF4A-Selective Inhibitor, Blocks Tumor Growth in Receptor Tyrosine Kinase Driven Tumors

    doi: 10.3389/fonc.2021.766298

    Figure Lengend Snippet: Zotatifin translationally downregulates FGFR1/2 and HER2 in RTK-driven tumors. (A) Schematic of RTK-stimulated PI3K/AKT and RAS/ERK pathways that activate eIF4A. (B) Frequency of HER2 and FGFR1/2 dysregulation in different cancer types. (C) Western blot analysis of RTKs and cyclin D1 levels following 24 h treatment with zotatifin or DMSO in HER2- or FGFR1/2-driven tumor models. β-actin or α-tubulin serve as loading controls. (D) Polysome profiling and mRNA distribution in MDA-MB-361 breast cancer cells treated for 3 h with 20 nM zotatifin compared to DMSO. mRNA levels of HER2 are monitored in polysome fractions. GAPDH serves as a control. (E) Luciferase reporter assay with gene constructs containing 5’-UTRs of FGFR1/2, HER2 or TUBA in HEK293 cells following 4 h of dose-dependent treatment with zotatifin. (F) Left, list of genes of which their 5’-UTR sequences were cloned into luciferase reporter constructs. Right, measured IC 50 values (nM) for inhibition of expression following 4 h treatment with zotatifin.

    Article Snippet: Primary antibodies: AKT CST#2920; AKT CST#9272; p-AKT S473 CST#4060; HER2 CST#2248; rpS6 CST#2317; rpS6 CST#2217; p-rpS6 S240/244 CST#5364; p-rpS6 S235/236 CST#4858; p70S6k CST#2708; p-p70S6k T389 CST#9234; ERK1/2 CST#4696; p-ERK1/2 T202/Y204 CST#4370; eIF4B CST#13088; p-eIF4B S406 CST#8151; p-eIF4B S422 CST#3591; 4EBP CST#9644; p-4EBP S65 CST#9456; PDCD4 CST#9535; Beta actin CST#3700; Beta tubulin CST#86298; PRAS40 CST#2691; p-PRAS40 T246 CST#13175; ER alpha CST#8644; FGFR2 CST#11835; HER3 #12708; p-HER3 #2842; FoxO3a #2497; p-FoxO1 (T24)/FoxO3a (T32) #9464 (Cell Signaling Technology, MA, USA), Cyclin D1 #241R-45 (MilliporeSigma, CA, USA), GAPDH (Santa Cruz Biotechnology, TX, USA).

    Techniques: Western Blot, Luciferase, Reporter Assay, Construct, Clone Assay, Inhibition, Expressing

    Zotatifin downregulates RTK levels induced by PI3K/AKT inhibition feedback in vitro . (A, B) SK-BR-3 HER2 amp cell line treated with zotatifin and Ipatasertib (AKTi); (A) Proliferation and apoptosis induction curves following 72 h of single agent or combination treatments (see also <xref ref-type= Figures 4B, C ). (B) Western blots analysis following 48 h treatment with single agents or combinations. (C) JIMT-1 HER2 amp cell line treated with zotatifin and Alpelisib (PIK3CAi) for 24 h; (D) MFM-223 FGFR2 amp cell line treated with zotatifin and Ipatasertib (AKTi) for 24 h; β-actin or GAPDH serve as loading controls. Quantification of p-HER3 Y1289 protein levels is shown for each condition. Concentrations of drugs used are indicated on top of each blot: Ipa., Ipatasertib (µM); Alp., Alpelisib (µM); zotatifin (nM); D, DMSO. " width="100%" height="100%">

    Journal: Frontiers in Oncology

    Article Title: Zotatifin, an eIF4A-Selective Inhibitor, Blocks Tumor Growth in Receptor Tyrosine Kinase Driven Tumors

    doi: 10.3389/fonc.2021.766298

    Figure Lengend Snippet: Zotatifin downregulates RTK levels induced by PI3K/AKT inhibition feedback in vitro . (A, B) SK-BR-3 HER2 amp cell line treated with zotatifin and Ipatasertib (AKTi); (A) Proliferation and apoptosis induction curves following 72 h of single agent or combination treatments (see also Figures 4B, C ). (B) Western blots analysis following 48 h treatment with single agents or combinations. (C) JIMT-1 HER2 amp cell line treated with zotatifin and Alpelisib (PIK3CAi) for 24 h; (D) MFM-223 FGFR2 amp cell line treated with zotatifin and Ipatasertib (AKTi) for 24 h; β-actin or GAPDH serve as loading controls. Quantification of p-HER3 Y1289 protein levels is shown for each condition. Concentrations of drugs used are indicated on top of each blot: Ipa., Ipatasertib (µM); Alp., Alpelisib (µM); zotatifin (nM); D, DMSO.

    Article Snippet: Primary antibodies: AKT CST#2920; AKT CST#9272; p-AKT S473 CST#4060; HER2 CST#2248; rpS6 CST#2317; rpS6 CST#2217; p-rpS6 S240/244 CST#5364; p-rpS6 S235/236 CST#4858; p70S6k CST#2708; p-p70S6k T389 CST#9234; ERK1/2 CST#4696; p-ERK1/2 T202/Y204 CST#4370; eIF4B CST#13088; p-eIF4B S406 CST#8151; p-eIF4B S422 CST#3591; 4EBP CST#9644; p-4EBP S65 CST#9456; PDCD4 CST#9535; Beta actin CST#3700; Beta tubulin CST#86298; PRAS40 CST#2691; p-PRAS40 T246 CST#13175; ER alpha CST#8644; FGFR2 CST#11835; HER3 #12708; p-HER3 #2842; FoxO3a #2497; p-FoxO1 (T24)/FoxO3a (T32) #9464 (Cell Signaling Technology, MA, USA), Cyclin D1 #241R-45 (MilliporeSigma, CA, USA), GAPDH (Santa Cruz Biotechnology, TX, USA).

    Techniques: Inhibition, In Vitro, Western Blot

    TRIB3 and HER2 expression exhibit a positive correlation in breast cancer. ( a ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in a panel of breast cancer cell lines (RNA seq data were obtained from the GSE48216 dataset). ( n = 56; p and R were calculated using Spearman’s correlation coefficient). ( b ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in breast cancer patients (RNA seq data were obtained from the TGCA Breast cancer dataset using the UCSC Xena platform) ( n = 1247; p and R were calculated using the Spearman correlation coefficient). ( c ) Correlation between TRIB3 protein levels (as determined by TRIB3 immunostaining and the subsequent assignment of the corresponding H value) with respect to HER2 levels (pathologically classified as 0-1-2-3 according to the level of expression of this receptor) in a tissue microarray that was generated with samples that were derived from luminal breast cancer patients ( n = 159; p was calculated using the Spearman’s correlation coefficient).

    Journal: Cancers

    Article Title: The Pseudokinase TRIB3 Negatively Regulates the HER2 Receptor Pathway and Is a Biomarker of Good Prognosis in Luminal Breast Cancer

    doi: 10.3390/cancers13215307

    Figure Lengend Snippet: TRIB3 and HER2 expression exhibit a positive correlation in breast cancer. ( a ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in a panel of breast cancer cell lines (RNA seq data were obtained from the GSE48216 dataset). ( n = 56; p and R were calculated using Spearman’s correlation coefficient). ( b ) Bioinformatic analysis of the correlation between TRIB3 and HER2 mRNA levels in breast cancer patients (RNA seq data were obtained from the TGCA Breast cancer dataset using the UCSC Xena platform) ( n = 1247; p and R were calculated using the Spearman correlation coefficient). ( c ) Correlation between TRIB3 protein levels (as determined by TRIB3 immunostaining and the subsequent assignment of the corresponding H value) with respect to HER2 levels (pathologically classified as 0-1-2-3 according to the level of expression of this receptor) in a tissue microarray that was generated with samples that were derived from luminal breast cancer patients ( n = 159; p was calculated using the Spearman’s correlation coefficient).

    Article Snippet: The membranes were then probed with the following primary antibodies: anti-HER2 (1:1000; Cell signaling, #2248, Danvers, MA, USA), anti-TRIB3 (1:1000; Abcam, #ab75846, Cambridge, UK), anti-pAKT S473 (1:1000; Cell signaling, #9271, Danvers, MA, USA), anti-pAKT T308 (1:1000; Cell signalling #9275, Danvers, MA, USA), anti-AKT (1:1000; Cell signaling, #9272, Danvers, MA, USA), anti-actin (1:4000; Sigma; A5441, St. Louis, MO, USA), anti-tubulin (1:4000; Sigma; T9026, St. Louis, MO, USA), or anti-HSP90 (1:2000; Sigma; SAB4200812, St. Louis, MO, USA).

    Techniques: Expressing, RNA Sequencing Assay, Immunostaining, Microarray, Generated, Derivative Assay

    TRIB3 and HER2 mutually regulate their levels. ( a ) The effect of HER2 silencing (siHER2) or transfection with a control siRNA (siC) on TRIB3 levels of BT474 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to siC ( n = 5; * p < 0.05 by using the Wilcoxon test). ( b ) The effect of HER2 overexpression (+HER2) or transfection with an empty vector (Φ) on TRIB3 levels of MCF7 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to the empty vector-transfected cells (Φ) ( n = 5; * p < 0.05 by using the Wilcoxon test). ( c ) Effect of TRIB3 silencing on HER2 mRNA (as determined by qRT-PCR) levels of BT474 cells. The data correspond to HER2 mRNA normalized with respect to the loading control (GAPDH) and are expressed as the mean fold change ± SEM with respect the shC condition ( n = 3). ( d ) The effect of TRIB3 stable knock-down on HER2 protein (as determined by Western blot) of MDA-MB-361, AU565, and BT474 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (β-ACTIN) and are expressed as the mean fold change ± SEM with respect to the shC condition ( n = 7; * p < 0.05 by using the Wilcoxon test). ( e ) The effect of TRIB3 silencing and incubation with the protein synthesis inhibitor cycloheximide (10 µM, CHX) on HER2 protein levels of BT474 cells at different timepoints. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to 0 h timepoint ( n = 3; * p < 0.05 with respect to shC cells; using the two-way ANOVA test). ( f ) The effect of treatment with Lapatinib (4 μM, 48 h) or vehicle (veh, DMSO) on the number of BT474 shC and shTRIB3 cells (as estimated by crystal violet staining). The data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 5; * p < 0.05 with respect to shC cells; using the two-way ANOVA test). ( g ) The effect of treatment with the AKT X inhibitor (10 μM, 48 h) or vehicle (veh, H 2 O) on the number of BT474 shC and shTRIB3 cells (as estimated by crystal violet staining). Data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 6; * p < 0.05, with respect to the shC cells; using the two-way ANOVA test).

    Journal: Cancers

    Article Title: The Pseudokinase TRIB3 Negatively Regulates the HER2 Receptor Pathway and Is a Biomarker of Good Prognosis in Luminal Breast Cancer

    doi: 10.3390/cancers13215307

    Figure Lengend Snippet: TRIB3 and HER2 mutually regulate their levels. ( a ) The effect of HER2 silencing (siHER2) or transfection with a control siRNA (siC) on TRIB3 levels of BT474 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to siC ( n = 5; * p < 0.05 by using the Wilcoxon test). ( b ) The effect of HER2 overexpression (+HER2) or transfection with an empty vector (Φ) on TRIB3 levels of MCF7 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to the empty vector-transfected cells (Φ) ( n = 5; * p < 0.05 by using the Wilcoxon test). ( c ) Effect of TRIB3 silencing on HER2 mRNA (as determined by qRT-PCR) levels of BT474 cells. The data correspond to HER2 mRNA normalized with respect to the loading control (GAPDH) and are expressed as the mean fold change ± SEM with respect the shC condition ( n = 3). ( d ) The effect of TRIB3 stable knock-down on HER2 protein (as determined by Western blot) of MDA-MB-361, AU565, and BT474 cells. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (β-ACTIN) and are expressed as the mean fold change ± SEM with respect to the shC condition ( n = 7; * p < 0.05 by using the Wilcoxon test). ( e ) The effect of TRIB3 silencing and incubation with the protein synthesis inhibitor cycloheximide (10 µM, CHX) on HER2 protein levels of BT474 cells at different timepoints. Left panel: The Western blot images of a representative experiment are shown. Right panel: The data correspond to the optical density values in arbitrary units for each experimental condition normalized with respect to the loading control (α-TUBULIN) and are expressed as the mean fold change ± SEM with respect to 0 h timepoint ( n = 3; * p < 0.05 with respect to shC cells; using the two-way ANOVA test). ( f ) The effect of treatment with Lapatinib (4 μM, 48 h) or vehicle (veh, DMSO) on the number of BT474 shC and shTRIB3 cells (as estimated by crystal violet staining). The data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 5; * p < 0.05 with respect to shC cells; using the two-way ANOVA test). ( g ) The effect of treatment with the AKT X inhibitor (10 μM, 48 h) or vehicle (veh, H 2 O) on the number of BT474 shC and shTRIB3 cells (as estimated by crystal violet staining). Data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 6; * p < 0.05, with respect to the shC cells; using the two-way ANOVA test).

    Article Snippet: The membranes were then probed with the following primary antibodies: anti-HER2 (1:1000; Cell signaling, #2248, Danvers, MA, USA), anti-TRIB3 (1:1000; Abcam, #ab75846, Cambridge, UK), anti-pAKT S473 (1:1000; Cell signaling, #9271, Danvers, MA, USA), anti-pAKT T308 (1:1000; Cell signalling #9275, Danvers, MA, USA), anti-AKT (1:1000; Cell signaling, #9272, Danvers, MA, USA), anti-actin (1:4000; Sigma; A5441, St. Louis, MO, USA), anti-tubulin (1:4000; Sigma; T9026, St. Louis, MO, USA), or anti-HSP90 (1:2000; Sigma; SAB4200812, St. Louis, MO, USA).

    Techniques: Transfection, Western Blot, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Incubation, Staining

    High TRIB3 levels correlate with better response to therapy in both HER2- and HER2+ luminal breast cancer. ( a ) The bioinformatic analysis of the association between TRIB3 mRNA levels and complete pathological response to endocrine therapies in ER-positive luminal A (left panel) and luminal B (right panel) breast cancer patients. The receiver operating characteristic (ROC) curves are shown. The area under the curve (AUC), p value, false positive rate (FPR), and true positive rate (TPR). ( b , c ) The correlation between TRIB3 protein levels (as determined by TRIB3 immunostaining) and recurrence after treatment with chemotherapy in a TMA of luminal breast cancer patients. Note that all patients had been initially treated with endocrine therapy. The data correspond to the H-value quantification of TRIB3 levels in the primary tumors and are expressed as the mean H value ± SEM for each set of patients. Panels b and c show information relative to all of the patients that were subjected to chemotherapy irrespective of HER2 status (panel B, n = 150) or separated according to HER2 expression (Panel c, n = 32 for HER2+ and n = 118 for HER2-). ** p < 0.01 and * p < 0.05 with respect to non-recurrent patients by using a T-test with Welch correction. ( d – g ) The effect of TRIB3 silencing (shTRIB3) and treatment with Doxorubicin ( d , e ) or Cisplatin ( f , g ) on the number of BT474 ( e , f ) and MCF7 ( d , g ) cells (as estimated by crystal violet staining). The data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 3–5; ** p < 0.01 and * p < 0.05 with respect to shC cells; using the two-way ANOVA test).

    Journal: Cancers

    Article Title: The Pseudokinase TRIB3 Negatively Regulates the HER2 Receptor Pathway and Is a Biomarker of Good Prognosis in Luminal Breast Cancer

    doi: 10.3390/cancers13215307

    Figure Lengend Snippet: High TRIB3 levels correlate with better response to therapy in both HER2- and HER2+ luminal breast cancer. ( a ) The bioinformatic analysis of the association between TRIB3 mRNA levels and complete pathological response to endocrine therapies in ER-positive luminal A (left panel) and luminal B (right panel) breast cancer patients. The receiver operating characteristic (ROC) curves are shown. The area under the curve (AUC), p value, false positive rate (FPR), and true positive rate (TPR). ( b , c ) The correlation between TRIB3 protein levels (as determined by TRIB3 immunostaining) and recurrence after treatment with chemotherapy in a TMA of luminal breast cancer patients. Note that all patients had been initially treated with endocrine therapy. The data correspond to the H-value quantification of TRIB3 levels in the primary tumors and are expressed as the mean H value ± SEM for each set of patients. Panels b and c show information relative to all of the patients that were subjected to chemotherapy irrespective of HER2 status (panel B, n = 150) or separated according to HER2 expression (Panel c, n = 32 for HER2+ and n = 118 for HER2-). ** p < 0.01 and * p < 0.05 with respect to non-recurrent patients by using a T-test with Welch correction. ( d – g ) The effect of TRIB3 silencing (shTRIB3) and treatment with Doxorubicin ( d , e ) or Cisplatin ( f , g ) on the number of BT474 ( e , f ) and MCF7 ( d , g ) cells (as estimated by crystal violet staining). The data correspond to the absorbance at 570 nm and are expressed as the mean fold change ± SEM with respect to veh-treated shC cells ( n = 3–5; ** p < 0.01 and * p < 0.05 with respect to shC cells; using the two-way ANOVA test).

    Article Snippet: The membranes were then probed with the following primary antibodies: anti-HER2 (1:1000; Cell signaling, #2248, Danvers, MA, USA), anti-TRIB3 (1:1000; Abcam, #ab75846, Cambridge, UK), anti-pAKT S473 (1:1000; Cell signaling, #9271, Danvers, MA, USA), anti-pAKT T308 (1:1000; Cell signalling #9275, Danvers, MA, USA), anti-AKT (1:1000; Cell signaling, #9272, Danvers, MA, USA), anti-actin (1:4000; Sigma; A5441, St. Louis, MO, USA), anti-tubulin (1:4000; Sigma; T9026, St. Louis, MO, USA), or anti-HSP90 (1:2000; Sigma; SAB4200812, St. Louis, MO, USA).

    Techniques: Immunostaining, Expressing, Staining

    A BT474 cells were treated with Leptin and AG1478 alone or in combination for 24 h. Cell lysates were immunoblotted with indicated antibodies. B BT474 cells were treated with Leptin and AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMed1, pHER2, and tHER2. Actin was used as loading control. C Bar graph shows fold change in protein expression in ( A ) and ( B ). D Immunoblot analysis of MCF7-vector and MCF7-Her2 O/E cells for indicated proteins. E , F MCF7-HER2 O/E cells were treated with Leptin, AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMED1, pHER2, and tHER2. Actin was used as loading control. Bar graph shows the ratio of MED1 and actin. G MCF7 and BT474 cells were treated as in ( A ) and ( B ), and subjected to immunofluorescence analysis of pMED1. Scale bar, 10 µm. H MCF7 cells were treated as indicated for 24 h and subjected to ChIP assay using Med1 antibody. The purified DNA was analyzed by using specific primer for ER promoter. ** p < 0.005, * p < 0.05. Data are means ± SD from three experiments.

    Journal: NPJ Breast Cancer

    Article Title: Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen by coordinating a crosstalk between Med1, miR205 and ErbB

    doi: 10.1038/s41523-021-00314-9

    Figure Lengend Snippet: A BT474 cells were treated with Leptin and AG1478 alone or in combination for 24 h. Cell lysates were immunoblotted with indicated antibodies. B BT474 cells were treated with Leptin and AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMed1, pHER2, and tHER2. Actin was used as loading control. C Bar graph shows fold change in protein expression in ( A ) and ( B ). D Immunoblot analysis of MCF7-vector and MCF7-Her2 O/E cells for indicated proteins. E , F MCF7-HER2 O/E cells were treated with Leptin, AG825 alone or in combination for 24 h. Cell lysates were immunoblotted for pMED1, pHER2, and tHER2. Actin was used as loading control. Bar graph shows the ratio of MED1 and actin. G MCF7 and BT474 cells were treated as in ( A ) and ( B ), and subjected to immunofluorescence analysis of pMED1. Scale bar, 10 µm. H MCF7 cells were treated as indicated for 24 h and subjected to ChIP assay using Med1 antibody. The purified DNA was analyzed by using specific primer for ER promoter. ** p < 0.005, * p < 0.05. Data are means ± SD from three experiments.

    Article Snippet: Antibodies for phosphorylated ER-ser 118 (2511; 1:500 dilution), ER-ser 167 (64508; 1:500 dilution), ERK (9102; 1:1000 dilution), phosphorylated-ERK (9101; 1:1000 dilution), EGFR (2232; 1:1000 dilution), phosphorylated EGFR (3777; 1:1000 dilution), Her2 (2248; 1:1000 dilution), phosphorylated Her2 (2247; 1:1000 dilution), c Myc (5605; 1:1000 dilution), cyclin D1 (2978; 1:1000 dilution), TFF1 (15571; 1:1000 dilution) were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Expressing, Western Blot, Plasmid Preparation, Immunofluorescence, Purification

    A Western blot and quantification of the levels of HER2, EGFR, and beta-actin in breast cancer cell lines, newly established cell lines, their corresponding PDX-derived organoids and PDX tumors. Expression of HER2 and EGFR for MC-BR-BTY-0019 cell line, organoid, and PDX was normalized to beta-actin and then SKBR3. Expression of HER2 and EGFR for MC-BR-BTY-0006 cell line, organoid, and PDX was normalized to beta-actin and then MDA-MB-231. All blots are derived from the same experiment and were processed in parallel. B MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with HER2 and DAPI and images were taken at ×100. Scale bar represents 10 μm. C MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with EGFR and DAPI and images were taken at ×100. Scale bar represents 10 μm. D 10,000 cells/well of MC-BR-BTY-0019 cell line were seeded in 96-well plates in triplicates, treated with indicated concentrations of lapatinib for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = 438.531 nM. E 10,000 cells/well of MC-BR-BTY-0006 cell line were seeded in 96-well plates in triplicates, treated with paclitaxel at indicated concentrations for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = Indeterminate. F , G PDX tumors were collected when the tumor achieved 10–20 mm in diameter, and primary breast cancer cells were isolated after dissociation of tumor tissue from mouse cells, as previously described . MC-BR-BTY-0019 PDX-derived organoids were generated by seeding 10,000 cells of the primary breast cancer single-cell suspension per well in Nanoculture plates in triplicates and treated with indicated concentrations of lapatinib for 5 days (IC 50 = 979.49 nM). MC-BR-BTY-0006 PDX-derived organoids were treated with indicated concentrations of paclitaxel for 5 days (IC 50 = Indeterminate). Organoid viability was measured using 3D Cell TiterGlo viability assay, normalized to DMSO, and plotted using GraphPad PRISM software. Error bars represent SEM.

    Journal: NPJ Breast Cancer

    Article Title: Establishment and characterization of immortalized human breast cancer cell lines from breast cancer patient-derived xenografts (PDX)

    doi: 10.1038/s41523-021-00285-x

    Figure Lengend Snippet: A Western blot and quantification of the levels of HER2, EGFR, and beta-actin in breast cancer cell lines, newly established cell lines, their corresponding PDX-derived organoids and PDX tumors. Expression of HER2 and EGFR for MC-BR-BTY-0019 cell line, organoid, and PDX was normalized to beta-actin and then SKBR3. Expression of HER2 and EGFR for MC-BR-BTY-0006 cell line, organoid, and PDX was normalized to beta-actin and then MDA-MB-231. All blots are derived from the same experiment and were processed in parallel. B MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with HER2 and DAPI and images were taken at ×100. Scale bar represents 10 μm. C MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were stained with EGFR and DAPI and images were taken at ×100. Scale bar represents 10 μm. D 10,000 cells/well of MC-BR-BTY-0019 cell line were seeded in 96-well plates in triplicates, treated with indicated concentrations of lapatinib for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = 438.531 nM. E 10,000 cells/well of MC-BR-BTY-0006 cell line were seeded in 96-well plates in triplicates, treated with paclitaxel at indicated concentrations for 48 h, and cell viability was measured by MTS assay and normalized to DMSO. IC 50 = Indeterminate. F , G PDX tumors were collected when the tumor achieved 10–20 mm in diameter, and primary breast cancer cells were isolated after dissociation of tumor tissue from mouse cells, as previously described . MC-BR-BTY-0019 PDX-derived organoids were generated by seeding 10,000 cells of the primary breast cancer single-cell suspension per well in Nanoculture plates in triplicates and treated with indicated concentrations of lapatinib for 5 days (IC 50 = 979.49 nM). MC-BR-BTY-0006 PDX-derived organoids were treated with indicated concentrations of paclitaxel for 5 days (IC 50 = Indeterminate). Organoid viability was measured using 3D Cell TiterGlo viability assay, normalized to DMSO, and plotted using GraphPad PRISM software. Error bars represent SEM.

    Article Snippet: Primary antibodies against beta-Actin (#4970; 1:1000), HER2/ErbB2 (#2248; 1:200) and EGFR (#4267; 1:1000) were purchased from Cell Signaling.

    Techniques: Western Blot, Derivative Assay, Expressing, Staining, MTS Assay, Isolation, Generated, Viability Assay, Software

    A , B Pathology staining of HE, Ki67, ER, PR, EGFR, and HER2 was performed in both PDX tumor tissue and CDX tumor formed from MC-BR-BTY-0019 cell line ( A ) and MC-BR-BTY-0006 ( B ). Images taken at ×200; Scale bar represents 200 μm. C Detailed description of the pathology staining features of tumor tissue of the PDX and CDX is summarized for MC-BR-BTY-0019 and MC-BR-BTY-0006. D MC-BR-BTY-0019 CDX in vivo tumors were formed by injecting 2 million cells of MC-BR-BTY-0019 cell line into 10 NSG mice, which were randomized when tumor achieved 50–100 mm 3 in volume, and treated with either control ( n = 5, vehicle, 5 days/week) or lapatinib ( n = 5, 50 mg/kg, 5 days/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. ** p < 0.01, 2 tailed Student’s t -test comparing vehicle to drug treatment. E MC-BR-BTY-0019 PDX tumor was formed by injecting earlier passage of PDX tumor into flank of 15 NSG mice, 13 mice with similar size of tumors were randomized when tumor achieved 50–100 mm 3 in volume and treated with either control ( n = 6, vehicle, 5 days/week) or lapatinib ( n = 7, 50 mg/kg, 5 days/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. ** p < 0.01, 2 tailed Student’s t -test comparing vehicle to drug treatment. F MC-BR-BTY-0006 CDX in vivo tumors were formed by injecting 5 million cells of MC-BR-BTY-0006 cell line into 10 NSG mice, which were randomized when the tumor reached 50–100 mm 3 in volume, and treated with either control ( n = 5, vehicle, 2 times/week) or paclitaxel ( n = 5, 12.5 mg/kg, 2 times/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. p -value = ns, 2 tailed Student’s t -test comparing vehicle to drug treatment. G MC-BR-BTY-0006 PDX tumor was formed by injecting earlier passage of PDX tumor into flank of 15 NSG mice, 12 mice with similar size of tumor were randomized when tumor reached 50–100 mm 3 in volume, and treated with either control ( n = 6, vehicle, 2 times/week) or paclitaxel ( n = 6, 12.5 mg/kg, 2 times/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. p -value = ns, 2 tailed Student’s t -test comparing vehicle to drug treatment.

    Journal: NPJ Breast Cancer

    Article Title: Establishment and characterization of immortalized human breast cancer cell lines from breast cancer patient-derived xenografts (PDX)

    doi: 10.1038/s41523-021-00285-x

    Figure Lengend Snippet: A , B Pathology staining of HE, Ki67, ER, PR, EGFR, and HER2 was performed in both PDX tumor tissue and CDX tumor formed from MC-BR-BTY-0019 cell line ( A ) and MC-BR-BTY-0006 ( B ). Images taken at ×200; Scale bar represents 200 μm. C Detailed description of the pathology staining features of tumor tissue of the PDX and CDX is summarized for MC-BR-BTY-0019 and MC-BR-BTY-0006. D MC-BR-BTY-0019 CDX in vivo tumors were formed by injecting 2 million cells of MC-BR-BTY-0019 cell line into 10 NSG mice, which were randomized when tumor achieved 50–100 mm 3 in volume, and treated with either control ( n = 5, vehicle, 5 days/week) or lapatinib ( n = 5, 50 mg/kg, 5 days/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. ** p < 0.01, 2 tailed Student’s t -test comparing vehicle to drug treatment. E MC-BR-BTY-0019 PDX tumor was formed by injecting earlier passage of PDX tumor into flank of 15 NSG mice, 13 mice with similar size of tumors were randomized when tumor achieved 50–100 mm 3 in volume and treated with either control ( n = 6, vehicle, 5 days/week) or lapatinib ( n = 7, 50 mg/kg, 5 days/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. ** p < 0.01, 2 tailed Student’s t -test comparing vehicle to drug treatment. F MC-BR-BTY-0006 CDX in vivo tumors were formed by injecting 5 million cells of MC-BR-BTY-0006 cell line into 10 NSG mice, which were randomized when the tumor reached 50–100 mm 3 in volume, and treated with either control ( n = 5, vehicle, 2 times/week) or paclitaxel ( n = 5, 12.5 mg/kg, 2 times/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. p -value = ns, 2 tailed Student’s t -test comparing vehicle to drug treatment. G MC-BR-BTY-0006 PDX tumor was formed by injecting earlier passage of PDX tumor into flank of 15 NSG mice, 12 mice with similar size of tumor were randomized when tumor reached 50–100 mm 3 in volume, and treated with either control ( n = 6, vehicle, 2 times/week) or paclitaxel ( n = 6, 12.5 mg/kg, 2 times/week) for 3 weeks. Tumor volume was measured at indicated time points and plotted using GraphPad PRISM software. Error bars represent SEM. p -value = ns, 2 tailed Student’s t -test comparing vehicle to drug treatment.

    Article Snippet: Primary antibodies against beta-Actin (#4970; 1:1000), HER2/ErbB2 (#2248; 1:200) and EGFR (#4267; 1:1000) were purchased from Cell Signaling.

    Techniques: Staining, In Vivo, Software

    A Oncogenic mutations in five cancer genes were detected in all three MC-BR-BTY-0006 tumor samples. Pathogenic loss-of-function somatic SNVs were detected in one allele of each of the genes TP53, PTEN, and BRCA2 as well as the other allele (loss of heterozygosity, or LOH). Thus, loss of function of both copies of tumor-suppressor genes TP53, PTEN, and BRCA2 could be inferred. RB1 loss of heterozygosity (LOH) and amplification of MYC were other oncogenic events observed. B Oncogenic mutations in eight cancer genes were detected in all three MC-BR-BTY-0019 tumor samples. A different pathogenic loss-of-function mutation in TP53 for cell line, but was again observed alongside LOH. So, loss of function of both copies can be inferred for TP53 as well as for three other tumor suppressor genes, CDKN2A, the adjacent CDKN2B, and MAP2K4, due to homozygous deletion (HomDel). Mutations were also observed in PIK3R1 and ARID1A. The mutations affect sites in regions frequently altered in cancer. Amplifications of TERT and ERBB2, or HER2, were also detected, with particularly high amplification for ERBB2. C Of the somatic SNV/INDELs detected in the patient tumor tissue samples, proportions detected in PDXs and cell lines were high (≥72%) for both patients. Of those mutations detected in PDXs, proportions detected in cell lines were also high (79%, 67%). D Changes in ploidy by sample type pair. For each patient, the absolute differences were nearly 1 copy from tissue to PDX with a Tissue→PDX increase observed for MC-BR-BTY-0006 and a Tissue→PDX decrease observed for MC-BR-BTY-0019. Despite these changes, the profiles of within-sample relative copy numbers for Tissue and PDX still showed similarity as the correlation of copy number was still 0.70 or greater. Slight decreases in ploidy were observed from PDX to cell line for both patients.

    Journal: NPJ Breast Cancer

    Article Title: Establishment and characterization of immortalized human breast cancer cell lines from breast cancer patient-derived xenografts (PDX)

    doi: 10.1038/s41523-021-00285-x

    Figure Lengend Snippet: A Oncogenic mutations in five cancer genes were detected in all three MC-BR-BTY-0006 tumor samples. Pathogenic loss-of-function somatic SNVs were detected in one allele of each of the genes TP53, PTEN, and BRCA2 as well as the other allele (loss of heterozygosity, or LOH). Thus, loss of function of both copies of tumor-suppressor genes TP53, PTEN, and BRCA2 could be inferred. RB1 loss of heterozygosity (LOH) and amplification of MYC were other oncogenic events observed. B Oncogenic mutations in eight cancer genes were detected in all three MC-BR-BTY-0019 tumor samples. A different pathogenic loss-of-function mutation in TP53 for cell line, but was again observed alongside LOH. So, loss of function of both copies can be inferred for TP53 as well as for three other tumor suppressor genes, CDKN2A, the adjacent CDKN2B, and MAP2K4, due to homozygous deletion (HomDel). Mutations were also observed in PIK3R1 and ARID1A. The mutations affect sites in regions frequently altered in cancer. Amplifications of TERT and ERBB2, or HER2, were also detected, with particularly high amplification for ERBB2. C Of the somatic SNV/INDELs detected in the patient tumor tissue samples, proportions detected in PDXs and cell lines were high (≥72%) for both patients. Of those mutations detected in PDXs, proportions detected in cell lines were also high (79%, 67%). D Changes in ploidy by sample type pair. For each patient, the absolute differences were nearly 1 copy from tissue to PDX with a Tissue→PDX increase observed for MC-BR-BTY-0006 and a Tissue→PDX decrease observed for MC-BR-BTY-0019. Despite these changes, the profiles of within-sample relative copy numbers for Tissue and PDX still showed similarity as the correlation of copy number was still 0.70 or greater. Slight decreases in ploidy were observed from PDX to cell line for both patients.

    Article Snippet: Primary antibodies against beta-Actin (#4970; 1:1000), HER2/ErbB2 (#2248; 1:200) and EGFR (#4267; 1:1000) were purchased from Cell Signaling.

    Techniques: Amplification, Mutagenesis

    (A and B) ARPE-19 cells were treated with 0.1 mM or 0.5 mM H 2 O 2 for the indicated times. (A) Cell death was analyzed by flow cytometry using propidium iodide staining. (B) The cell lysates were immunoblotted with the indicated antibodies. (C) ARPE-19 cells were treated with 0.5 mM H 2 O 2 in the presence or absence of the autophagy inhibitors 3-methyladenine (3-MA, 10 µM) and bafilomycin A1 (BafA1, 100 nM). The cell lysates were immunoblotted with the indicated antibodies. Cont, control. (B and C) LC3-II/-I ratio was quantified by densitometric analyses. (D) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 or autophagy modulators (inducer: rapamycin, 100 nM, or inhibitor: BafA1, 100 nM) for 6 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Scale bars = 10 µm. (A and D) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t-test. *P < 0.05, **P < 0.01.

    Journal: Molecules and Cells

    Article Title: PARP1 Impedes SIRT1-Mediated Autophagy during Degeneration of the Retinal Pigment Epithelium under Oxidative Stress

    doi: 10.14348/molcells.2020.0078

    Figure Lengend Snippet: (A and B) ARPE-19 cells were treated with 0.1 mM or 0.5 mM H 2 O 2 for the indicated times. (A) Cell death was analyzed by flow cytometry using propidium iodide staining. (B) The cell lysates were immunoblotted with the indicated antibodies. (C) ARPE-19 cells were treated with 0.5 mM H 2 O 2 in the presence or absence of the autophagy inhibitors 3-methyladenine (3-MA, 10 µM) and bafilomycin A1 (BafA1, 100 nM). The cell lysates were immunoblotted with the indicated antibodies. Cont, control. (B and C) LC3-II/-I ratio was quantified by densitometric analyses. (D) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 or autophagy modulators (inducer: rapamycin, 100 nM, or inhibitor: BafA1, 100 nM) for 6 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Scale bars = 10 µm. (A and D) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t-test. *P < 0.05, **P < 0.01.

    Article Snippet: Bafilomycin A1, hydrogen peroxide, 3-methyl adenine, β-nicotinamide mononucleotide (β-NMN), chloroquine (CQ), wortmannin, sodium iodate (SI), 50% glutaraldehyde, rapamycin (RAPA), a rabbit anti-p62 antibody, and a mouse β-actin antibody were purchased from Sigma-Aldrich (USA); a horseradish peroxidase (HRP)-conjugated anti-mouse antibody, a HRP-conjugated anti-rabbit antibody, a fluorescein-conjugated anti-mouse antibody and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Thermo Fisher Scientific (USA); a mouse anti-PARP1 antibody was purchased from BD Biosciences (USA); rabbit anti-LC3, anti-lysosomal-associated membrane protein (LAMP1), anti-phospho mammalian target of rapamycin (mTOR) (serine 2248), anti-mTOR, and anti-acetyl p53 (lysine 382) antibodies were purchased from Cell Signaling Technology (USA); rabbit anti-AIF, mouse anti-p53, and mouse anti-RPE65 antibodies were purchased from Santa Cruz Biotechnology (USA); a mouse anti-PAR antibody was purchased from Enzo Life Sciences (USA) and Abcam (England); and olaparib was purchased from Selleck Chemicals (USA).

    Techniques: Flow Cytometry, Staining, Transfection

    (A) ARPE-19 cells were treated with 0.5 mM H 2 O 2 for the indicated times in the presence or absence of 10 µM olaparib (Ola). Veh, vehicle. (B and C) ARPE-19 cells were transfected with scrambled siRNA (scRNA) or ATG7 targeting siRNA for 48 h. Then, the cells were treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (B) for indicated times (C). (D and F) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (D) or pretreated with 100 µM rapamycin and then treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (F). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Cont, control. Scale bars = 10 µm. (E and F) ARPE-19 cells were pretreated with 100 µM rapamycin for 5 h and then treated with 0.5 mM H 2 O 2 for 1 h in the presence or absence of 10 µM olaparib. (A, B, C, and E) The cell lysates were immunoblotted with the indicated antibodies. The LC3-II/-I ratio and relative p62 levels were quantified by densitometric analyses (ImageJ software). (D and F) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001.

    Journal: Molecules and Cells

    Article Title: PARP1 Impedes SIRT1-Mediated Autophagy during Degeneration of the Retinal Pigment Epithelium under Oxidative Stress

    doi: 10.14348/molcells.2020.0078

    Figure Lengend Snippet: (A) ARPE-19 cells were treated with 0.5 mM H 2 O 2 for the indicated times in the presence or absence of 10 µM olaparib (Ola). Veh, vehicle. (B and C) ARPE-19 cells were transfected with scrambled siRNA (scRNA) or ATG7 targeting siRNA for 48 h. Then, the cells were treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (B) for indicated times (C). (D and F) ARPE-19 cells were transfected with RFP-GFP-LC3 and seeded on poly-D-lysine-coated coverslips. Next, the cells were treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (D) or pretreated with 100 µM rapamycin and then treated with 0.5 mM H 2 O 2 for 6 h in the presence or absence of 10 µM olaparib (F). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Red and yellow arrows indicate autolysosomes and autophagosomes, respectively. Cont, control. Scale bars = 10 µm. (E and F) ARPE-19 cells were pretreated with 100 µM rapamycin for 5 h and then treated with 0.5 mM H 2 O 2 for 1 h in the presence or absence of 10 µM olaparib. (A, B, C, and E) The cell lysates were immunoblotted with the indicated antibodies. The LC3-II/-I ratio and relative p62 levels were quantified by densitometric analyses (ImageJ software). (D and F) Quantified data are expressed as the mean ± SD from three independent biological replicates. Statistical analysis was performed by Student’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001.

    Article Snippet: Bafilomycin A1, hydrogen peroxide, 3-methyl adenine, β-nicotinamide mononucleotide (β-NMN), chloroquine (CQ), wortmannin, sodium iodate (SI), 50% glutaraldehyde, rapamycin (RAPA), a rabbit anti-p62 antibody, and a mouse β-actin antibody were purchased from Sigma-Aldrich (USA); a horseradish peroxidase (HRP)-conjugated anti-mouse antibody, a HRP-conjugated anti-rabbit antibody, a fluorescein-conjugated anti-mouse antibody and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Thermo Fisher Scientific (USA); a mouse anti-PARP1 antibody was purchased from BD Biosciences (USA); rabbit anti-LC3, anti-lysosomal-associated membrane protein (LAMP1), anti-phospho mammalian target of rapamycin (mTOR) (serine 2248), anti-mTOR, and anti-acetyl p53 (lysine 382) antibodies were purchased from Cell Signaling Technology (USA); rabbit anti-AIF, mouse anti-p53, and mouse anti-RPE65 antibodies were purchased from Santa Cruz Biotechnology (USA); a mouse anti-PAR antibody was purchased from Enzo Life Sciences (USA) and Abcam (England); and olaparib was purchased from Selleck Chemicals (USA).

    Techniques: Transfection, Staining, Software