Structured Review

Millipore erbb2
MM-121 in combination with trastuzumab significantly inhibits in vivo growth of tumor xenografts established from BT474-HR20 trastuzumab-resistant breast cancer cells. BT474-HR20 cells were s.c. injected into nude mice to establish tumor xenografts. The tumor-bearing mice (n = 5) received i.p. injections of PBS, trastuzumab, MM-121, or trastuzumab plus MM-121 as described in the Materials and Methods. After 6 treatments, the mice were euthanized at day 36 post injection of tumor cells, and all tumors were excised for histology and IHC analysis. A , The graphs show the tumor growth curves. Bars , SD. The combinations of MM-121 and trastuzumab significantly inhibited tumor growth as compared to control or single Ab treatment. B , Data show the representative tumors with hematoxylin and eosin (H E) staining and IHC analysis of <t>erbB2</t> and erbB3. The residual tumor cells obtained from combinatorial treatments retained similar expression levels of erbB2/erbB3 receptors on the cell membrane.
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1) Product Images from "The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells"

Article Title: The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-12-134

MM-121 in combination with trastuzumab significantly inhibits in vivo growth of tumor xenografts established from BT474-HR20 trastuzumab-resistant breast cancer cells. BT474-HR20 cells were s.c. injected into nude mice to establish tumor xenografts. The tumor-bearing mice (n = 5) received i.p. injections of PBS, trastuzumab, MM-121, or trastuzumab plus MM-121 as described in the Materials and Methods. After 6 treatments, the mice were euthanized at day 36 post injection of tumor cells, and all tumors were excised for histology and IHC analysis. A , The graphs show the tumor growth curves. Bars , SD. The combinations of MM-121 and trastuzumab significantly inhibited tumor growth as compared to control or single Ab treatment. B , Data show the representative tumors with hematoxylin and eosin (H E) staining and IHC analysis of erbB2 and erbB3. The residual tumor cells obtained from combinatorial treatments retained similar expression levels of erbB2/erbB3 receptors on the cell membrane.
Figure Legend Snippet: MM-121 in combination with trastuzumab significantly inhibits in vivo growth of tumor xenografts established from BT474-HR20 trastuzumab-resistant breast cancer cells. BT474-HR20 cells were s.c. injected into nude mice to establish tumor xenografts. The tumor-bearing mice (n = 5) received i.p. injections of PBS, trastuzumab, MM-121, or trastuzumab plus MM-121 as described in the Materials and Methods. After 6 treatments, the mice were euthanized at day 36 post injection of tumor cells, and all tumors were excised for histology and IHC analysis. A , The graphs show the tumor growth curves. Bars , SD. The combinations of MM-121 and trastuzumab significantly inhibited tumor growth as compared to control or single Ab treatment. B , Data show the representative tumors with hematoxylin and eosin (H E) staining and IHC analysis of erbB2 and erbB3. The residual tumor cells obtained from combinatorial treatments retained similar expression levels of erbB2/erbB3 receptors on the cell membrane.

Techniques Used: In Vivo, Injection, Mouse Assay, Immunohistochemistry, Staining, Expressing

The addition of MM-121 enhances trastuzumab-induced cell cycle G1 arrest in erbB2+ breast cancer cell lines. SKBR3 and BT474 cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. A , Half of the cells were collected and subjected to western blot analyses with specific antibodies directed against E2F-1, Cyclin D1, p27 kip1 , or β-actin. The densitometry analyses of E2F-1, Cyclin D1, and p27 kip1 signals were shown underneath, and the arbitrary numbers indicate the intensities of each sample relative to controls, defined as 1.0. B C , The other half of the cells were collected for analysis of cell cycle distributions by flow cytometry as described in the Materials and Methods. Data show a representative of three independent experiments.
Figure Legend Snippet: The addition of MM-121 enhances trastuzumab-induced cell cycle G1 arrest in erbB2+ breast cancer cell lines. SKBR3 and BT474 cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. A , Half of the cells were collected and subjected to western blot analyses with specific antibodies directed against E2F-1, Cyclin D1, p27 kip1 , or β-actin. The densitometry analyses of E2F-1, Cyclin D1, and p27 kip1 signals were shown underneath, and the arbitrary numbers indicate the intensities of each sample relative to controls, defined as 1.0. B C , The other half of the cells were collected for analysis of cell cycle distributions by flow cytometry as described in the Materials and Methods. Data show a representative of three independent experiments.

Techniques Used: Western Blot, Flow Cytometry, Cytometry

MM-121 enhances trastuzumab-mediated inactivation of Akt and growth inhibition in two erbB2+ breast cancer cell lines. A , SKBR3 and BT474 breast cancer cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. Cells were collected and subjected to western blot analyses of P-erbB2, erbB2, P-erbB3, erbB3, P-Akt, Akt, P-MAPK, MAPK, or β-actin. B , SKBR3 and BT474 cells were plated onto 96-well plates and incubated at 37°C with 5% CO2. After 24 hrs, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of trastuzumab in the absence (Trast) or presence (Trast + MM-121) of MM-121 (10 μg/ml) for another 72 hrs. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS. Bars , SD. Data show a representative of three independent experiments.
Figure Legend Snippet: MM-121 enhances trastuzumab-mediated inactivation of Akt and growth inhibition in two erbB2+ breast cancer cell lines. A , SKBR3 and BT474 breast cancer cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. Cells were collected and subjected to western blot analyses of P-erbB2, erbB2, P-erbB3, erbB3, P-Akt, Akt, P-MAPK, MAPK, or β-actin. B , SKBR3 and BT474 cells were plated onto 96-well plates and incubated at 37°C with 5% CO2. After 24 hrs, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of trastuzumab in the absence (Trast) or presence (Trast + MM-121) of MM-121 (10 μg/ml) for another 72 hrs. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS. Bars , SD. Data show a representative of three independent experiments.

Techniques Used: Inhibition, Western Blot, Incubation

2) Product Images from "Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer"

Article Title: Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr3563

MM-121 significantly enhances paclitaxel-mediated anti-proliferative/anti-survival effects on breast cancer cell lines with expression of both erbB2 and erbB3. (A) SKBR3.neo1, SKBR3.B3.1, or SKBR3.B3.2 cells were plated onto 96-well plates and incubated at 37°C with 5% CO 2 . After 24 h, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of paclitaxel in the absence (paclitaxel) or presence (MM-121 + pac) of MM-121 (10 μg/ml) for another 72 h. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS). Bars represent SD. Data are representative of three independent experiments. (B) The same cells were untreated or treated with MM-121 (10 μg/ml) for 24 or 48 h. Cells were collected and subjected to western blot analyses of phosphorylated (P)-erbB2, erbB2, P-erbB3, erbB3, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK, or β-actin.
Figure Legend Snippet: MM-121 significantly enhances paclitaxel-mediated anti-proliferative/anti-survival effects on breast cancer cell lines with expression of both erbB2 and erbB3. (A) SKBR3.neo1, SKBR3.B3.1, or SKBR3.B3.2 cells were plated onto 96-well plates and incubated at 37°C with 5% CO 2 . After 24 h, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of paclitaxel in the absence (paclitaxel) or presence (MM-121 + pac) of MM-121 (10 μg/ml) for another 72 h. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS). Bars represent SD. Data are representative of three independent experiments. (B) The same cells were untreated or treated with MM-121 (10 μg/ml) for 24 or 48 h. Cells were collected and subjected to western blot analyses of phosphorylated (P)-erbB2, erbB2, P-erbB3, erbB3, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK, or β-actin.

Techniques Used: Expressing, Incubation, Western Blot

3) Product Images from "Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane"

Article Title: Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane

Journal: Oncology Letters

doi: 10.3892/ol.2015.4043

HK II dissociation from mitochondria is increased in ErbB2-overexpressing breast cancer cells. (A) The intact mitochondrial fractions from 231V and 231ErbB2 cells and (B) BT474 scramble and BT474 siErbB2 cells were isolated and treated with 3-BrPA for
Figure Legend Snippet: HK II dissociation from mitochondria is increased in ErbB2-overexpressing breast cancer cells. (A) The intact mitochondrial fractions from 231V and 231ErbB2 cells and (B) BT474 scramble and BT474 siErbB2 cells were isolated and treated with 3-BrPA for

Techniques Used: Isolation

Overexpression of ErbB2 in breast cancer cells increases mitochondrial localization and activity of HK II. (A) Overexpression of ErbB2 upregulated the expression of HK II. Whole cell lysates were collected and for western blotting analysis, and β-actin
Figure Legend Snippet: Overexpression of ErbB2 in breast cancer cells increases mitochondrial localization and activity of HK II. (A) Overexpression of ErbB2 upregulated the expression of HK II. Whole cell lysates were collected and for western blotting analysis, and β-actin

Techniques Used: Over Expression, Activity Assay, Expressing, Western Blot

3-BrPA has a greater inhibitory effect in ErbB2-overexpressing breast cancer cells than control cells in nude mice. Pre-established 231V or 231ErbB2 tumor xenografts were treated with control (phosphate-buffered saline), 3-BrPA (10 mg/kg, twice/week for
Figure Legend Snippet: 3-BrPA has a greater inhibitory effect in ErbB2-overexpressing breast cancer cells than control cells in nude mice. Pre-established 231V or 231ErbB2 tumor xenografts were treated with control (phosphate-buffered saline), 3-BrPA (10 mg/kg, twice/week for

Techniques Used: Mouse Assay

Breast cancer cells with a high expression of ErbB2 are more sensitive to glucose depletion than ErbB2-negative cells. (A) Overexpression of ErbB2 in MCF7 and MDA-MB-231 breast cancer cells, and siRNA knockdown of ErbB2 in BT474 breast cancer cells. Cells
Figure Legend Snippet: Breast cancer cells with a high expression of ErbB2 are more sensitive to glucose depletion than ErbB2-negative cells. (A) Overexpression of ErbB2 in MCF7 and MDA-MB-231 breast cancer cells, and siRNA knockdown of ErbB2 in BT474 breast cancer cells. Cells

Techniques Used: Expressing, Over Expression, Multiple Displacement Amplification

Overexpression of ErbB2 decreased the mitochondria membrane potential following 3-BrPA treatment, inducing apoptosis. (A) The mitochondria membrane potential (MMP) was measured in 231V, 231ErbB2, BT474 scramble and BT474 siErbB2 cells following treatment
Figure Legend Snippet: Overexpression of ErbB2 decreased the mitochondria membrane potential following 3-BrPA treatment, inducing apoptosis. (A) The mitochondria membrane potential (MMP) was measured in 231V, 231ErbB2, BT474 scramble and BT474 siErbB2 cells following treatment

Techniques Used: Over Expression

4) Product Images from "Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism"

Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

Journal: Nature communications

doi: 10.1038/ncomms2236

Translocation of ErbB2 into mitochondria is kinase dependent. (A) The mitochondrial fractions and whole cell lysates were isolated and Western blotting was performed on MDA-MB-435ErbB2 (435eb), MDA-MB-435ErbB2V695E (435VE) and MDA-MB-435ErbB2K753M (435KM) cells. MtHSP70 and α-Tubulin were loading controls. (B) Mitochondrial fractions and whole cell lysates were isolated and Western Blotting was performed to detect mtErbB2 in mitochondria following the treatment of MCF7ErbB2 cells with ErbB2 inhibitor AG825 at 10 uM or control (DMSO) for 24 h; and with Heregulin β1 (HRG) at 10 ng/ml or control (PBS) for 24 h (upper). Whole cell lysates were subjected to Western Blotting to examine the phosphorylation status of ErbB2 and the total ErbB2. α-Tubulin was loading control (lower). (C) MCF7 cells were treated with and without HRG at 10 ng/ml for 24 h. The mitochondrial fraction and the whole cell lysate were isolated for Western blotting. Prohibitin and α-Tubulin were loading controls. (D) Left, mitochondrial proteins were isolated from SKBR3 cells and immunoprecipitated with anti-ErbB2 or anti-Cox II antibodies. The immunoprecipitates were analyzed by Western blotting with anti-ErbB2 and anti-Cox II antibodies. IgG was negative control. Right, mitochondrial proteins were isolated from 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells and immunoprecipited with Cox II antibody or negative control IgG. Samples were loaded on a SDS–PAGE followed by Western Blotting analysis with Cox II antibody to show the Cox II protein level, and with P-Y20 antibody to show the tyrosine phosphorylation level on Cox II. (E) Cytochrome c oxidase activities were measured after the cells were treated with or without a cytochrome c oxidase inhibitor Potassium cyanide (KCN) in 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells. The relative activities of Cox II were calculated relative to the Cox II activity of 231V cells. (F) 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells were treated with or without Taxol at 80 nM for 24 h. Cytosolic, mitochondrial fractions and whole cell lysates were analyzed by Western blotting with cytochrome c antibodies. α-Tubulin and mtHSP70 were cytosol and mitochondrial markers and loading controls. Columns , mean of three independent experiments; bars , SE.*, P
Figure Legend Snippet: Translocation of ErbB2 into mitochondria is kinase dependent. (A) The mitochondrial fractions and whole cell lysates were isolated and Western blotting was performed on MDA-MB-435ErbB2 (435eb), MDA-MB-435ErbB2V695E (435VE) and MDA-MB-435ErbB2K753M (435KM) cells. MtHSP70 and α-Tubulin were loading controls. (B) Mitochondrial fractions and whole cell lysates were isolated and Western Blotting was performed to detect mtErbB2 in mitochondria following the treatment of MCF7ErbB2 cells with ErbB2 inhibitor AG825 at 10 uM or control (DMSO) for 24 h; and with Heregulin β1 (HRG) at 10 ng/ml or control (PBS) for 24 h (upper). Whole cell lysates were subjected to Western Blotting to examine the phosphorylation status of ErbB2 and the total ErbB2. α-Tubulin was loading control (lower). (C) MCF7 cells were treated with and without HRG at 10 ng/ml for 24 h. The mitochondrial fraction and the whole cell lysate were isolated for Western blotting. Prohibitin and α-Tubulin were loading controls. (D) Left, mitochondrial proteins were isolated from SKBR3 cells and immunoprecipitated with anti-ErbB2 or anti-Cox II antibodies. The immunoprecipitates were analyzed by Western blotting with anti-ErbB2 and anti-Cox II antibodies. IgG was negative control. Right, mitochondrial proteins were isolated from 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells and immunoprecipited with Cox II antibody or negative control IgG. Samples were loaded on a SDS–PAGE followed by Western Blotting analysis with Cox II antibody to show the Cox II protein level, and with P-Y20 antibody to show the tyrosine phosphorylation level on Cox II. (E) Cytochrome c oxidase activities were measured after the cells were treated with or without a cytochrome c oxidase inhibitor Potassium cyanide (KCN) in 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells. The relative activities of Cox II were calculated relative to the Cox II activity of 231V cells. (F) 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells were treated with or without Taxol at 80 nM for 24 h. Cytosolic, mitochondrial fractions and whole cell lysates were analyzed by Western blotting with cytochrome c antibodies. α-Tubulin and mtHSP70 were cytosol and mitochondrial markers and loading controls. Columns , mean of three independent experiments; bars , SE.*, P

Techniques Used: Translocation Assay, Isolation, Western Blot, Multiple Displacement Amplification, Immunoprecipitation, Negative Control, SDS Page, Activity Assay

Localization of ErbB2 in mitochondria. (A) Cytosolic, nuclear, mitochondrial, and plasma membrane proteins were isolated and subjected to SDS-PAGE followed by probing with indicated antibodies. Two exogenous ErbB2 overexpressing breast cancer cell lines MCF7/ErbB2 and MDA-MB-231 (left), and two natural ErbB2-positive breast cancer cell lines SKBR3 and BT474 (right) were used for Western Blotting. VDAC1 and prohibitin were mitochondrial markers; Integrin β1 and IGF1Rα were plasma membrane markers; α-Tubulin and ERK were cytoplasmic markers; KDEL was an ER marker; EEA1 was an early endosomes marker; Golgi complex was a marker for the detection of Golgi; LAMP2 was an lysosome marker and Lamin B1 was a nucleus marker. (B) MCF7 cells, mouse heart and liver tissues, and ErbB2 positive and negative breast cancer patient samples were analyzed by Western blotting. (C) Co-localization of ErbB2 and mitochondria. Mitochondria were stained with Mitotracker-Green in ErbB2 transfected MCF7 cells. The cells were fixed and incubated with antibodies against ErbB2 (mouse), followed by incubation of monoclonal mouse Anti–Cy3 antibody (red). Images were analyzed with Nikon NIS-Elements AR software. Green: mitochondria; Red: ErbB2; Yellow: Co-localization of ErbB2 and mitochondria. The lower panel contains images with a higher magnification. Scale bars: 20 μm. (D) Localization of ErbB2 inside mitochondria. Intact mitochondria of SKBR3 cells were isolated and treated with Protease K at 0.5 mg/ml for 15 min at room temperature. 1% Triton was added into the reaction and 2 mM PMSF was added to stop the reaction. Each reaction was started with an equal amount/volume of mitochondria lysate. Mitochondrial lysate then was loaded onto 8% SDS PAGE followed by blotting with anti-ErbB2, anti-BCL-2 and anti-Prohibitin antibodies.
Figure Legend Snippet: Localization of ErbB2 in mitochondria. (A) Cytosolic, nuclear, mitochondrial, and plasma membrane proteins were isolated and subjected to SDS-PAGE followed by probing with indicated antibodies. Two exogenous ErbB2 overexpressing breast cancer cell lines MCF7/ErbB2 and MDA-MB-231 (left), and two natural ErbB2-positive breast cancer cell lines SKBR3 and BT474 (right) were used for Western Blotting. VDAC1 and prohibitin were mitochondrial markers; Integrin β1 and IGF1Rα were plasma membrane markers; α-Tubulin and ERK were cytoplasmic markers; KDEL was an ER marker; EEA1 was an early endosomes marker; Golgi complex was a marker for the detection of Golgi; LAMP2 was an lysosome marker and Lamin B1 was a nucleus marker. (B) MCF7 cells, mouse heart and liver tissues, and ErbB2 positive and negative breast cancer patient samples were analyzed by Western blotting. (C) Co-localization of ErbB2 and mitochondria. Mitochondria were stained with Mitotracker-Green in ErbB2 transfected MCF7 cells. The cells were fixed and incubated with antibodies against ErbB2 (mouse), followed by incubation of monoclonal mouse Anti–Cy3 antibody (red). Images were analyzed with Nikon NIS-Elements AR software. Green: mitochondria; Red: ErbB2; Yellow: Co-localization of ErbB2 and mitochondria. The lower panel contains images with a higher magnification. Scale bars: 20 μm. (D) Localization of ErbB2 inside mitochondria. Intact mitochondria of SKBR3 cells were isolated and treated with Protease K at 0.5 mg/ml for 15 min at room temperature. 1% Triton was added into the reaction and 2 mM PMSF was added to stop the reaction. Each reaction was started with an equal amount/volume of mitochondria lysate. Mitochondrial lysate then was loaded onto 8% SDS PAGE followed by blotting with anti-ErbB2, anti-BCL-2 and anti-Prohibitin antibodies.

Techniques Used: Isolation, SDS Page, Multiple Displacement Amplification, Western Blot, Marker, Staining, Transfection, Incubation, Software

Analysis of the mechanisms of mtErbB2 mitochondrial localization. (A) MCF7 breast cancer cells were transfected with plasmids encoding either GFP alone, or GFP fused ErbB2 fragments GFP-646–689, GFP-623–645 or GFP-623–689. Cells were cultured and the florescent imaging was done as described under the “Methods.” Scale bars: 20 μm. (B) MDA-MB-231 cells were transfected with wild-type ErbB2 and ErbB2ΔMTS vectors and mitochondrial proteins were extracted. ErbB2 expression was measured by Western blotting analysis; α-Tubulin and mtHSP70 were loading controls. (C) Mitochondrial proteins were isolated from MDA-MB-231ErbB2 cells and immunoprecipitated with ErbB2 antibody. The immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top left). Mitochondrial proteins from the same cells were precipitated with mtHSP70 antibody and the immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top right). IgG was used as a negative control. Isolated mitochondrial proteins (input) were loaded as a positive control. Similar results were obtained using another breast cancer cell line, SKBR3 (bottom). (D) siRNA specific to mtHSP70 was transfected into MDA-MB-231ErbB2 cells. The cytoplasmic fraction (Cyto), mitochondrial fraction (Mito) and whole cell lysate (WCL) were separated for western blotting analysis (left). Cytochrome c oxidase subunit II and α-Tubulin were makers and loading controls for the mitochondrial fraction and the cytoplasmic fraction, respectively. The relative protein amounts of ErbB2 and mtHSP70 were calculated by determining the intensity of the protein bands followed by the normalization with loading controls (right). Experiments were repeated three times. Columns, mean of three independent experiments; bars, SE.
Figure Legend Snippet: Analysis of the mechanisms of mtErbB2 mitochondrial localization. (A) MCF7 breast cancer cells were transfected with plasmids encoding either GFP alone, or GFP fused ErbB2 fragments GFP-646–689, GFP-623–645 or GFP-623–689. Cells were cultured and the florescent imaging was done as described under the “Methods.” Scale bars: 20 μm. (B) MDA-MB-231 cells were transfected with wild-type ErbB2 and ErbB2ΔMTS vectors and mitochondrial proteins were extracted. ErbB2 expression was measured by Western blotting analysis; α-Tubulin and mtHSP70 were loading controls. (C) Mitochondrial proteins were isolated from MDA-MB-231ErbB2 cells and immunoprecipitated with ErbB2 antibody. The immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top left). Mitochondrial proteins from the same cells were precipitated with mtHSP70 antibody and the immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top right). IgG was used as a negative control. Isolated mitochondrial proteins (input) were loaded as a positive control. Similar results were obtained using another breast cancer cell line, SKBR3 (bottom). (D) siRNA specific to mtHSP70 was transfected into MDA-MB-231ErbB2 cells. The cytoplasmic fraction (Cyto), mitochondrial fraction (Mito) and whole cell lysate (WCL) were separated for western blotting analysis (left). Cytochrome c oxidase subunit II and α-Tubulin were makers and loading controls for the mitochondrial fraction and the cytoplasmic fraction, respectively. The relative protein amounts of ErbB2 and mtHSP70 were calculated by determining the intensity of the protein bands followed by the normalization with loading controls (right). Experiments were repeated three times. Columns, mean of three independent experiments; bars, SE.

Techniques Used: Transfection, Cell Culture, Imaging, Multiple Displacement Amplification, Expressing, Western Blot, Isolation, Immunoprecipitation, Negative Control, Positive Control

Translocation of ErbB2 into mitochondria contributes to trastuzumab resistance. (A) mtErbB2 is elevated in trastuzumab-resistant cancer cells. Mitochondrial proteins and whole cell lysates of BT474 and BT474 trastuzumab-resistant cells (BT474 HCP R) were isolated and analyzed by Western blotting. ATP Synthase and α-Tubulin were used as loading controls. (B) BT474 cells were treated with trastuzumab (HCP) at 10 ug/ml for 24 h and 48 h followed by the separation of cytosolic and mitochondrial fractions. Proteins from the cytoplasm, mitochondria and whole cell lysates were loaded onto gels and analyzed by Western blotting. mtHSP70 and α-Tubulin were used as loading controls (left). The relative protein amount of ErbB2 in the cellular fractions was calculated by detecting the intensity of the protein bands followed by normalization with loading controls (right). The experiments were repeated for three times. (C) mtErbB2-overexpressing cells are more resistant to trastuzumab. 231ErbB2WT, 231ErbB2Mito, 231ErbB2ΔMTS, 231ErbB2MitoKM and 231ErbB2WT cells transfected by siRNA to mtHSP70 were treated with trastuzumab at 100 ug/ml for 48 h and 72 h. The cell growth inhibition ratios were detected by a CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit. Columns, mean of three independent experiments; bars, SE. *, P
Figure Legend Snippet: Translocation of ErbB2 into mitochondria contributes to trastuzumab resistance. (A) mtErbB2 is elevated in trastuzumab-resistant cancer cells. Mitochondrial proteins and whole cell lysates of BT474 and BT474 trastuzumab-resistant cells (BT474 HCP R) were isolated and analyzed by Western blotting. ATP Synthase and α-Tubulin were used as loading controls. (B) BT474 cells were treated with trastuzumab (HCP) at 10 ug/ml for 24 h and 48 h followed by the separation of cytosolic and mitochondrial fractions. Proteins from the cytoplasm, mitochondria and whole cell lysates were loaded onto gels and analyzed by Western blotting. mtHSP70 and α-Tubulin were used as loading controls (left). The relative protein amount of ErbB2 in the cellular fractions was calculated by detecting the intensity of the protein bands followed by normalization with loading controls (right). The experiments were repeated for three times. (C) mtErbB2-overexpressing cells are more resistant to trastuzumab. 231ErbB2WT, 231ErbB2Mito, 231ErbB2ΔMTS, 231ErbB2MitoKM and 231ErbB2WT cells transfected by siRNA to mtHSP70 were treated with trastuzumab at 100 ug/ml for 48 h and 72 h. The cell growth inhibition ratios were detected by a CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit. Columns, mean of three independent experiments; bars, SE. *, P

Techniques Used: Translocation Assay, Isolation, Western Blot, Transfection, Inhibition, Proliferation Assay

5) Product Images from "Destabilization of ERBB2 transcripts by targeting 3? UTR mRNA associated HuR and histone deacetylase-6 (HDAC6)"

Article Title: Destabilization of ERBB2 transcripts by targeting 3? UTR mRNA associated HuR and histone deacetylase-6 (HDAC6)

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-07-2110

HDAC6-selective inhibitor ST71 fails to repress intracellular ERBB2 promoter activity but inhibits culture growth of ERBB2-positive SKBR3 cells. A. Dose-response comparison against ERBB2 promoter-reporting (luciferase expressing) MCF7/R06pGL-4 cells following
Figure Legend Snippet: HDAC6-selective inhibitor ST71 fails to repress intracellular ERBB2 promoter activity but inhibits culture growth of ERBB2-positive SKBR3 cells. A. Dose-response comparison against ERBB2 promoter-reporting (luciferase expressing) MCF7/R06pGL-4 cells following

Techniques Used: Activity Assay, Luciferase, Expressing

Association of cytosolic HuR with ERBB2 mRNA and HDAC enzymatic activity, and the co-precipitation and co-localization of HuR with HDAC6 in SKBR3 cells. A. Immunoprecipitation of cytoplasmic HuR by specific (or isotype control) antibody followed by RT-PCR
Figure Legend Snippet: Association of cytosolic HuR with ERBB2 mRNA and HDAC enzymatic activity, and the co-precipitation and co-localization of HuR with HDAC6 in SKBR3 cells. A. Immunoprecipitation of cytoplasmic HuR by specific (or isotype control) antibody followed by RT-PCR

Techniques Used: Activity Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

ERBB2 effects of pan-HDAC enzymatic inhibitors (HDACi) are independent of HuR levels and associated with transcript binding to HDAC6, but dissimilar to that of HDAC6 protein downregulation by siRNA. A. ERBB2 protein inhibition by 16–20 h of SKBR3
Figure Legend Snippet: ERBB2 effects of pan-HDAC enzymatic inhibitors (HDACi) are independent of HuR levels and associated with transcript binding to HDAC6, but dissimilar to that of HDAC6 protein downregulation by siRNA. A. ERBB2 protein inhibition by 16–20 h of SKBR3

Techniques Used: Binding Assay, Inhibition

HDAC6 knockdown offsets ERBB2 mRNA decay induced by pan-HDACi and HDAC6-selective inhibitors that comparably reduce SKBR3 ERBB2 mRNA and protein levels. A. Northern blot showing the influence of 3 h treatment with 0.5 μM LAQ824, 50 μM
Figure Legend Snippet: HDAC6 knockdown offsets ERBB2 mRNA decay induced by pan-HDACi and HDAC6-selective inhibitors that comparably reduce SKBR3 ERBB2 mRNA and protein levels. A. Northern blot showing the influence of 3 h treatment with 0.5 μM LAQ824, 50 μM

Techniques Used: Northern Blot

ERBB2 expression is regulated by the transcript stability factor, HuR, which binds a conserved U-rich element in the 3′UTR of ERBB2 mRNA. A. Near the poly-A tail of the ERBB2 3′ UTR is an evolutionarily conserved U-rich region (nt 465–505).
Figure Legend Snippet: ERBB2 expression is regulated by the transcript stability factor, HuR, which binds a conserved U-rich element in the 3′UTR of ERBB2 mRNA. A. Near the poly-A tail of the ERBB2 3′ UTR is an evolutionarily conserved U-rich region (nt 465–505).

Techniques Used: Expressing

6) Product Images from "Geldanamycin selectively targets the nascent form of ERBB3 for degradation"

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation

Journal: Cell Stress & Chaperones

doi: 10.1007/s12192-009-0166-1

The kinase domain of EGFR resembles ERBB3 with respect to the charge distribution in the HSP90 interface but is closer to ERBB2 in phylogeny. a Phylogenetic nearest neighbor analysis of ERBB kinase domains and their orthologs. Drosophila DER is added
Figure Legend Snippet: The kinase domain of EGFR resembles ERBB3 with respect to the charge distribution in the HSP90 interface but is closer to ERBB2 in phylogeny. a Phylogenetic nearest neighbor analysis of ERBB kinase domains and their orthologs. Drosophila DER is added

Techniques Used:

After geldanamycin treatment, receptor cross-linking increases substantially for cell surface ERBB2, but not for ERBB3. ERBB2 and ERBB3 were individually overexpressed in CHO cells. Following treatment with either DMSO or GA for 2 h at 37°C
Figure Legend Snippet: After geldanamycin treatment, receptor cross-linking increases substantially for cell surface ERBB2, but not for ERBB3. ERBB2 and ERBB3 were individually overexpressed in CHO cells. Following treatment with either DMSO or GA for 2 h at 37°C

Techniques Used:

Mature ERBB3 is not destabilized by GA. a Levels of cell surface ERBB2 and ERBB3 after GA treatment, relative to unchallenged turnover. MCF7 cells were cell-surface-biotinylated with 1 mg/ml NHS-biotin and subsequently treated with 3 µM
Figure Legend Snippet: Mature ERBB3 is not destabilized by GA. a Levels of cell surface ERBB2 and ERBB3 after GA treatment, relative to unchallenged turnover. MCF7 cells were cell-surface-biotinylated with 1 mg/ml NHS-biotin and subsequently treated with 3 µM

Techniques Used:

7) Product Images from "A novel luciferase based reporter system to monitor activation of the ErbB2/Her2/neu pathway non-invasively during radiotherapy"

Article Title: A novel luciferase based reporter system to monitor activation of the ErbB2/Her2/neu pathway non-invasively during radiotherapy

Journal: International journal of radiation oncology, biology, physics

doi: 10.1016/j.ijrobp.2010.08.001

Monitoring erbB2/Her2 reporter activity in cancer cells exposed to cytotoxic treatments.
Figure Legend Snippet: Monitoring erbB2/Her2 reporter activity in cancer cells exposed to cytotoxic treatments.

Techniques Used: Activity Assay

Non-invasive imaging of ErbB2 activation after irradiation. Mice (n=5) have been injected with 3×10 5 cells in each hind leg. When tumors reached a size of 7–8mm in diameter, the right hind leg was irradiated with a dose of 5Gy. Mice were
Figure Legend Snippet: Non-invasive imaging of ErbB2 activation after irradiation. Mice (n=5) have been injected with 3×10 5 cells in each hind leg. When tumors reached a size of 7–8mm in diameter, the right hind leg was irradiated with a dose of 5Gy. Mice were

Techniques Used: Imaging, Activation Assay, Irradiation, Mouse Assay, Injection

8) Product Images from "The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells"

Article Title: The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-12-134

MM-121 in combination with trastuzumab significantly inhibits in vivo growth of tumor xenografts established from BT474-HR20 trastuzumab-resistant breast cancer cells. BT474-HR20 cells were s.c. injected into nude mice to establish tumor xenografts. The tumor-bearing mice (n = 5) received i.p. injections of PBS, trastuzumab, MM-121, or trastuzumab plus MM-121 as described in the Materials and Methods. After 6 treatments, the mice were euthanized at day 36 post injection of tumor cells, and all tumors were excised for histology and IHC analysis. A , The graphs show the tumor growth curves. Bars , SD. The combinations of MM-121 and trastuzumab significantly inhibited tumor growth as compared to control or single Ab treatment. B , Data show the representative tumors with hematoxylin and eosin (H E) staining and IHC analysis of erbB2 and erbB3. The residual tumor cells obtained from combinatorial treatments retained similar expression levels of erbB2/erbB3 receptors on the cell membrane.
Figure Legend Snippet: MM-121 in combination with trastuzumab significantly inhibits in vivo growth of tumor xenografts established from BT474-HR20 trastuzumab-resistant breast cancer cells. BT474-HR20 cells were s.c. injected into nude mice to establish tumor xenografts. The tumor-bearing mice (n = 5) received i.p. injections of PBS, trastuzumab, MM-121, or trastuzumab plus MM-121 as described in the Materials and Methods. After 6 treatments, the mice were euthanized at day 36 post injection of tumor cells, and all tumors were excised for histology and IHC analysis. A , The graphs show the tumor growth curves. Bars , SD. The combinations of MM-121 and trastuzumab significantly inhibited tumor growth as compared to control or single Ab treatment. B , Data show the representative tumors with hematoxylin and eosin (H E) staining and IHC analysis of erbB2 and erbB3. The residual tumor cells obtained from combinatorial treatments retained similar expression levels of erbB2/erbB3 receptors on the cell membrane.

Techniques Used: In Vivo, Injection, Mouse Assay, Immunohistochemistry, Staining, Expressing

The addition of MM-121 enhances trastuzumab-induced cell cycle G1 arrest in erbB2+ breast cancer cell lines. SKBR3 and BT474 cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. A , Half of the cells were collected and subjected to western blot analyses with specific antibodies directed against E2F-1, Cyclin D1, p27 kip1 , or β-actin. The densitometry analyses of E2F-1, Cyclin D1, and p27 kip1 signals were shown underneath, and the arbitrary numbers indicate the intensities of each sample relative to controls, defined as 1.0. B C , The other half of the cells were collected for analysis of cell cycle distributions by flow cytometry as described in the Materials and Methods. Data show a representative of three independent experiments.
Figure Legend Snippet: The addition of MM-121 enhances trastuzumab-induced cell cycle G1 arrest in erbB2+ breast cancer cell lines. SKBR3 and BT474 cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. A , Half of the cells were collected and subjected to western blot analyses with specific antibodies directed against E2F-1, Cyclin D1, p27 kip1 , or β-actin. The densitometry analyses of E2F-1, Cyclin D1, and p27 kip1 signals were shown underneath, and the arbitrary numbers indicate the intensities of each sample relative to controls, defined as 1.0. B C , The other half of the cells were collected for analysis of cell cycle distributions by flow cytometry as described in the Materials and Methods. Data show a representative of three independent experiments.

Techniques Used: Western Blot, Flow Cytometry, Cytometry

MM-121 enhances trastuzumab-mediated inactivation of Akt and growth inhibition in two erbB2+ breast cancer cell lines. A , SKBR3 and BT474 breast cancer cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. Cells were collected and subjected to western blot analyses of P-erbB2, erbB2, P-erbB3, erbB3, P-Akt, Akt, P-MAPK, MAPK, or β-actin. B , SKBR3 and BT474 cells were plated onto 96-well plates and incubated at 37°C with 5% CO2. After 24 hrs, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of trastuzumab in the absence (Trast) or presence (Trast + MM-121) of MM-121 (10 μg/ml) for another 72 hrs. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS. Bars , SD. Data show a representative of three independent experiments.
Figure Legend Snippet: MM-121 enhances trastuzumab-mediated inactivation of Akt and growth inhibition in two erbB2+ breast cancer cell lines. A , SKBR3 and BT474 breast cancer cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. Cells were collected and subjected to western blot analyses of P-erbB2, erbB2, P-erbB3, erbB3, P-Akt, Akt, P-MAPK, MAPK, or β-actin. B , SKBR3 and BT474 cells were plated onto 96-well plates and incubated at 37°C with 5% CO2. After 24 hrs, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of trastuzumab in the absence (Trast) or presence (Trast + MM-121) of MM-121 (10 μg/ml) for another 72 hrs. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS. Bars , SD. Data show a representative of three independent experiments.

Techniques Used: Inhibition, Western Blot, Incubation

9) Product Images from "Mitochondrially targeted vitamin E succinate efficiently kills breast tumour-initiating cells in a complex II-dependent manner"

Article Title: Mitochondrially targeted vitamin E succinate efficiently kills breast tumour-initiating cells in a complex II-dependent manner

Journal: BMC Cancer

doi: 10.1186/s12885-015-1394-7

NeuTL and MCF7 spheres are a plausible model of TICs. Neu TL cells were cultured in serum-containing and sphere medium ( A ) and assessed for selected stemness genes by qPCR ( C ). MCF7 cells were cultured in adherent and ‘sphere’ medium ( B ) and assessed for selected stemness genes by qPCR ( D ). ( E ) NeuTL adherent and sphere cells were grafted s.c. in FVB/N c-neu mice (10 6 cells per animal) and tumour volume assessed using USI. The images on the right are representative USI scans of tumours taken on the given days (indicated by arrows in the graph on the left). ( F ) Sections of tumours were stained by H E for morphology, also showing regions of low and more differentiated cancer cells. ( G ) Tumour sections were evaluated for the level of erbB2 using WB and IHC. In all cases, the level of stemness genes in sphere cells was related to that in their adherent counterparts, set as 1. Data are mean values ± S.D. (n = 3). The symbol ‘*’ indicates statistically significant differences in the level of mRNA in adherent and sphere cells with p
Figure Legend Snippet: NeuTL and MCF7 spheres are a plausible model of TICs. Neu TL cells were cultured in serum-containing and sphere medium ( A ) and assessed for selected stemness genes by qPCR ( C ). MCF7 cells were cultured in adherent and ‘sphere’ medium ( B ) and assessed for selected stemness genes by qPCR ( D ). ( E ) NeuTL adherent and sphere cells were grafted s.c. in FVB/N c-neu mice (10 6 cells per animal) and tumour volume assessed using USI. The images on the right are representative USI scans of tumours taken on the given days (indicated by arrows in the graph on the left). ( F ) Sections of tumours were stained by H E for morphology, also showing regions of low and more differentiated cancer cells. ( G ) Tumour sections were evaluated for the level of erbB2 using WB and IHC. In all cases, the level of stemness genes in sphere cells was related to that in their adherent counterparts, set as 1. Data are mean values ± S.D. (n = 3). The symbol ‘*’ indicates statistically significant differences in the level of mRNA in adherent and sphere cells with p

Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Mouse Assay, Staining, Western Blot, Immunohistochemistry

10) Product Images from "Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer"

Article Title: Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr3563

MM-121 significantly enhances paclitaxel-mediated anti-proliferative/anti-survival effects on breast cancer cell lines with expression of both erbB2 and erbB3. (A) SKBR3.neo1, SKBR3.B3.1, or SKBR3.B3.2 cells were plated onto 96-well plates and incubated at 37°C with 5% CO 2 . After 24 h, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of paclitaxel in the absence (paclitaxel) or presence (MM-121 + pac) of MM-121 (10 μg/ml) for another 72 h. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS). Bars represent SD. Data are representative of three independent experiments. (B) The same cells were untreated or treated with MM-121 (10 μg/ml) for 24 or 48 h. Cells were collected and subjected to western blot analyses of phosphorylated (P)-erbB2, erbB2, P-erbB3, erbB3, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK, or β-actin.
Figure Legend Snippet: MM-121 significantly enhances paclitaxel-mediated anti-proliferative/anti-survival effects on breast cancer cell lines with expression of both erbB2 and erbB3. (A) SKBR3.neo1, SKBR3.B3.1, or SKBR3.B3.2 cells were plated onto 96-well plates and incubated at 37°C with 5% CO 2 . After 24 h, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of paclitaxel in the absence (paclitaxel) or presence (MM-121 + pac) of MM-121 (10 μg/ml) for another 72 h. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS). Bars represent SD. Data are representative of three independent experiments. (B) The same cells were untreated or treated with MM-121 (10 μg/ml) for 24 or 48 h. Cells were collected and subjected to western blot analyses of phosphorylated (P)-erbB2, erbB2, P-erbB3, erbB3, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK, or β-actin.

Techniques Used: Expressing, Incubation, Western Blot

11) Product Images from "miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism"

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S90530

Overexpression of miR-125b resensitizes doxorubicin resistant chondrosarcoma cells through the inhibition of glucose metabolism. Notes: ( A ) Overexpression of miR-125b resensitized doxorubicin resistant cells. Cells were transfected with pre-miR-125b for 48 hours and then were treated with doxorubicin at indicated concentrations for 48 hours, followed by the measurement of cell viability. ( B ) Western blotting experiments showed the overexpression of miR-125b in JJ012 doxorubicin resistant cells decreased the expressions of ErbB2, HK II, PDK1, and LDHA to the same levels as those of JJ012 parental cells. ( C ) Glucose uptake (left) and lactate product (right) results showed the overexpression of miR-125b in JJ012 doxorubicin resistant cells decreased the glucose metabolism to the same levels as those of JJ012 parental cells. ( D ) Exogenous overexpression of LDHA into miR-125b pretransfected JJ012 parental cells (left) showed resistant to doxorubicin treatments at the indicated concentrations for 48 hours (right). Columns, mean of three independent experiments; bars, SE. * P
Figure Legend Snippet: Overexpression of miR-125b resensitizes doxorubicin resistant chondrosarcoma cells through the inhibition of glucose metabolism. Notes: ( A ) Overexpression of miR-125b resensitized doxorubicin resistant cells. Cells were transfected with pre-miR-125b for 48 hours and then were treated with doxorubicin at indicated concentrations for 48 hours, followed by the measurement of cell viability. ( B ) Western blotting experiments showed the overexpression of miR-125b in JJ012 doxorubicin resistant cells decreased the expressions of ErbB2, HK II, PDK1, and LDHA to the same levels as those of JJ012 parental cells. ( C ) Glucose uptake (left) and lactate product (right) results showed the overexpression of miR-125b in JJ012 doxorubicin resistant cells decreased the glucose metabolism to the same levels as those of JJ012 parental cells. ( D ) Exogenous overexpression of LDHA into miR-125b pretransfected JJ012 parental cells (left) showed resistant to doxorubicin treatments at the indicated concentrations for 48 hours (right). Columns, mean of three independent experiments; bars, SE. * P

Techniques Used: Over Expression, Inhibition, Transfection, Western Blot

Restoration of ErbB2 in miR-125b overexpressing cells recovers the glucose metabolism and doxorubicin sensitivity. Notes: ( A ) JJ012 cells were transfected with 100 nM pre-miR-negative control and pre-miR-125b for 48 hours, followed by transfection with vector control and overexpression vector containing wild-type ErbB2 for 24 hours, and then cells were collected and prepared for Western blotting with antibody against ErbB2, HK II, PDK1, and LDHA. β-actin was used as a loading control. ( B ) JJ012 cells were transfected with pre-miR-125b and ErbB2 as described in ( A ), then the glucose uptake (left) and lactate product (right) were checked. ( C ) JJ012 cells were transfected with pre-miR-125b and ErbB2 as described in ( A ); cells were collected and replaced in 48-well plates for overnight, followed by doxorubicin treatments at the indicated concentrations for 48 hours. Cells were analyzed by cell viability assays. ( D ) The JJ012 cells were transfected with control siRNA or siErbB2 for 48 hours, and then cells were collected for Western blot analysis (middle) and the measurements of glucose uptake and lactate product. Columns, mean of three independent experiments; bars, SE. * P
Figure Legend Snippet: Restoration of ErbB2 in miR-125b overexpressing cells recovers the glucose metabolism and doxorubicin sensitivity. Notes: ( A ) JJ012 cells were transfected with 100 nM pre-miR-negative control and pre-miR-125b for 48 hours, followed by transfection with vector control and overexpression vector containing wild-type ErbB2 for 24 hours, and then cells were collected and prepared for Western blotting with antibody against ErbB2, HK II, PDK1, and LDHA. β-actin was used as a loading control. ( B ) JJ012 cells were transfected with pre-miR-125b and ErbB2 as described in ( A ), then the glucose uptake (left) and lactate product (right) were checked. ( C ) JJ012 cells were transfected with pre-miR-125b and ErbB2 as described in ( A ); cells were collected and replaced in 48-well plates for overnight, followed by doxorubicin treatments at the indicated concentrations for 48 hours. Cells were analyzed by cell viability assays. ( D ) The JJ012 cells were transfected with control siRNA or siErbB2 for 48 hours, and then cells were collected for Western blot analysis (middle) and the measurements of glucose uptake and lactate product. Columns, mean of three independent experiments; bars, SE. * P

Techniques Used: Transfection, Negative Control, Plasmid Preparation, Over Expression, Western Blot

ErbB2 is a direct target of miR-125b in chondrosarcoma. Notes: ( A ) JJ012 and ( B ) CH-2879 cells were transfected with 100 nM pre-miR-negative (Ctr), pre-miR-125b, or anti-miR-125b for 48 hours. Cell lysates were prepared for Western blotting with antibody against ErbB2 and EGFR. EGFR was used as negative control and β-actin was used as a loading control (left). JJ012 and CH-2879 cells were cotransfected with luciferase reporter plasmids with wild-type 3′-UTR of ErbB2 or mutant 3′-UTR of ErbB2 and pre-miR-125, or pre-miR-negative (control miR) by using Lipofectamine 2000 reagent. Forty-eight hours posttransfection, cells were harvested and lysed with passive lysis buffer. Luciferase activities were measured by a dual luciferase reporter assay. The pRL-TK vector was used as an internal control. The results were expressed as relative luciferase activity (firefly LUC/Renilla LUC) (right). Columns, mean of three independent experiments; bars, SE. * P
Figure Legend Snippet: ErbB2 is a direct target of miR-125b in chondrosarcoma. Notes: ( A ) JJ012 and ( B ) CH-2879 cells were transfected with 100 nM pre-miR-negative (Ctr), pre-miR-125b, or anti-miR-125b for 48 hours. Cell lysates were prepared for Western blotting with antibody against ErbB2 and EGFR. EGFR was used as negative control and β-actin was used as a loading control (left). JJ012 and CH-2879 cells were cotransfected with luciferase reporter plasmids with wild-type 3′-UTR of ErbB2 or mutant 3′-UTR of ErbB2 and pre-miR-125, or pre-miR-negative (control miR) by using Lipofectamine 2000 reagent. Forty-eight hours posttransfection, cells were harvested and lysed with passive lysis buffer. Luciferase activities were measured by a dual luciferase reporter assay. The pRL-TK vector was used as an internal control. The results were expressed as relative luciferase activity (firefly LUC/Renilla LUC) (right). Columns, mean of three independent experiments; bars, SE. * P

Techniques Used: Transfection, Western Blot, Negative Control, Luciferase, Mutagenesis, Lysis, Reporter Assay, Plasmid Preparation, Activity Assay

12) Product Images from "Increased erbB3 promotes erbB2/neu-driven mammary tumor proliferation and co-targeting of erbB2/erbB3 receptors exhibits potent inhibitory effects on breast cancer cells"

Article Title: Increased erbB3 promotes erbB2/neu-driven mammary tumor proliferation and co-targeting of erbB2/erbB3 receptors exhibits potent inhibitory effects on breast cancer cells

Journal: International Journal of Clinical and Experimental Pathology

doi:

Combinations of Ab105 and Herceptin decreased P-Akt levels and significantly induced growth inhibition in erbB2+ breast cancer cells. A. MDA-MB-453 and 435.eB1 breast cancer cells were cultured in DMEM/F12 medium containing 0.5% FBS or the same medium
Figure Legend Snippet: Combinations of Ab105 and Herceptin decreased P-Akt levels and significantly induced growth inhibition in erbB2+ breast cancer cells. A. MDA-MB-453 and 435.eB1 breast cancer cells were cultured in DMEM/F12 medium containing 0.5% FBS or the same medium

Techniques Used: Inhibition, Multiple Displacement Amplification, Cell Culture

Increased mouse erbB3 significantly promoted wt rat c- erbB2/neu -driven mammary tumor growth in vivo . (A) Mammary tumors obtained from the transgenic mice were evaluated by IHC analysis with specific Abs against erbB2, P-erbB2, erbB3, and P-erbB3. Representative
Figure Legend Snippet: Increased mouse erbB3 significantly promoted wt rat c- erbB2/neu -driven mammary tumor growth in vivo . (A) Mammary tumors obtained from the transgenic mice were evaluated by IHC analysis with specific Abs against erbB2, P-erbB2, erbB3, and P-erbB3. Representative

Techniques Used: In Vivo, Transgenic Assay, Mouse Assay, Immunohistochemistry

Herceptin did not recognize rat erbB2, whereas MM-121 bound to both human and mouse erbB3 and reduced the levels of P-erbB2, P-erbB3, and P-Akt in mouse mammary tumor cells. A. Human (SKBR3 and BT474) and mouse (85815 and 85819) breast/mammary tumor cell
Figure Legend Snippet: Herceptin did not recognize rat erbB2, whereas MM-121 bound to both human and mouse erbB3 and reduced the levels of P-erbB2, P-erbB3, and P-Akt in mouse mammary tumor cells. A. Human (SKBR3 and BT474) and mouse (85815 and 85819) breast/mammary tumor cell

Techniques Used:

Overexpression of erbB2 increased the levels of erbB3 protein, but not mRNA, in human breast cancer cells through a mechanism involving in miR-125a, miR-125b, and miR-205. A. Same amount of total lysates from MCF-7 and MCF-7/erbB2 cells were analyzed
Figure Legend Snippet: Overexpression of erbB2 increased the levels of erbB3 protein, but not mRNA, in human breast cancer cells through a mechanism involving in miR-125a, miR-125b, and miR-205. A. Same amount of total lysates from MCF-7 and MCF-7/erbB2 cells were analyzed

Techniques Used: Over Expression

Interactions between mouse erbB3 and rat erbB2 were discovered in the mammary tumors-derived from the wt rat c- erbB2/neu -transgenic mice. A. Two representative mammary tumors 81230 and 81231 were homogenized in the presence of Western blot lysis buffer.
Figure Legend Snippet: Interactions between mouse erbB3 and rat erbB2 were discovered in the mammary tumors-derived from the wt rat c- erbB2/neu -transgenic mice. A. Two representative mammary tumors 81230 and 81231 were homogenized in the presence of Western blot lysis buffer.

Techniques Used: Derivative Assay, Transgenic Assay, Mouse Assay, Western Blot, Lysis

13) Product Images from "PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage"

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

Journal: Oncogene

doi: 10.1038/onc.2010.153

PERK loss attenuates MMTV- Neu -driven mammary tumorigenesis in mice, but promotes spontaneous mammary tumor formation in aged mammary gland-specific PERK knockout mice (A) Kaplan-Meier analysis of tumor-free survival for cohorts of MMTV-Neu/PERK loxP/loxP (n=21) and MMTV-Neu/PERK Δ/Δ Δ/Δ (n=27) mice. (B) Hematoxylin and eosin staining demonstrating histology of control (Neu/PERK loxP/loxP ) and PERK knockout (Neu PERK Δ/Δ ) mammary gland tumors. (C) Western analysis for PERK, ErbB2, and eIF4E levels on whole protein extracts from control (Neu/PERK loxP/loxP ) and PERK knockout (Neu/PERK Δ/Δ ) mammary gland tumors or mammary gland from lactating PERK loxP/loxP dam (L10). (D) Nrf2 was precipitated from tumor lysates prepared from MMTV-Neu/PERK Δ/Δ or control mice and blotted for phospho-Thr and Nrf2. PERK expression was determined by immunoblot. (E) PERK excision delays development of Neu-driven hyperplastic lesions. Representative mammary glands from 9- to 14-months old control (Neu/PERK loxP/loxP ) and PERK knockout (Neu/PERK Δ/Δ ) mice revealing pre-malignant lesions are shown. (F) Hematoxylin and eosin staining on lungs from control (Neu/PERK loxP/loxP , n=24) and PERK knockout (Neu/PERK Δ/Δ , n=27) mice revealing metastatic lesions. (G) Troma-1 (cytokeratin-8) staining on lung specimens containing metastatic foci. LT=lung tissue; Met=metastasis; OL=overlay. (H) Hematoxylin and eosin staining for tumor histology and whole mount of hyperplastic lesions in mammary glands of PERK Δ/Δ aged females. (I) qRT-PCR for ErbB2 on genomic DNA from PERK Δ/Δ tumors and FISH analysis on paraffin sections from the same animals. Levels of ErbB2 in tumors were compared to matched spleen tissues.
Figure Legend Snippet: PERK loss attenuates MMTV- Neu -driven mammary tumorigenesis in mice, but promotes spontaneous mammary tumor formation in aged mammary gland-specific PERK knockout mice (A) Kaplan-Meier analysis of tumor-free survival for cohorts of MMTV-Neu/PERK loxP/loxP (n=21) and MMTV-Neu/PERK Δ/Δ Δ/Δ (n=27) mice. (B) Hematoxylin and eosin staining demonstrating histology of control (Neu/PERK loxP/loxP ) and PERK knockout (Neu PERK Δ/Δ ) mammary gland tumors. (C) Western analysis for PERK, ErbB2, and eIF4E levels on whole protein extracts from control (Neu/PERK loxP/loxP ) and PERK knockout (Neu/PERK Δ/Δ ) mammary gland tumors or mammary gland from lactating PERK loxP/loxP dam (L10). (D) Nrf2 was precipitated from tumor lysates prepared from MMTV-Neu/PERK Δ/Δ or control mice and blotted for phospho-Thr and Nrf2. PERK expression was determined by immunoblot. (E) PERK excision delays development of Neu-driven hyperplastic lesions. Representative mammary glands from 9- to 14-months old control (Neu/PERK loxP/loxP ) and PERK knockout (Neu/PERK Δ/Δ ) mice revealing pre-malignant lesions are shown. (F) Hematoxylin and eosin staining on lungs from control (Neu/PERK loxP/loxP , n=24) and PERK knockout (Neu/PERK Δ/Δ , n=27) mice revealing metastatic lesions. (G) Troma-1 (cytokeratin-8) staining on lung specimens containing metastatic foci. LT=lung tissue; Met=metastasis; OL=overlay. (H) Hematoxylin and eosin staining for tumor histology and whole mount of hyperplastic lesions in mammary glands of PERK Δ/Δ aged females. (I) qRT-PCR for ErbB2 on genomic DNA from PERK Δ/Δ tumors and FISH analysis on paraffin sections from the same animals. Levels of ErbB2 in tumors were compared to matched spleen tissues.

Techniques Used: Mouse Assay, Knock-Out, Staining, Western Blot, Expressing, Quantitative RT-PCR, Fluorescence In Situ Hybridization

PERK expression is maintained in cancer cells wherein it regulates tumor expansion in vivo (A) PERK protein levels were measured by immunoprecipitation (IP) followed by Western blot analysis in the following cell lines: MCF10A (1), MCF7 (2), T47D (3), MDA-MB231 (4), MDA-MB468 (5), TE3 (6), TE7 (7), KYSE 520 (8). (B) PERK protein levels following shRNA targeting of PERK. (C) Parental MDA-MB468 cell line, shPERK-transduced cells (shPERK), and shPERK-transduced cells reconstituted with mouse Myc-PERK (+mPERK) were treated with 2μg/ml tunicamycin for the indicated intervals. Western analysis for ATF4, CHOP, or β-actin. (D) Volume of orthotopic tumors formed from the mouse mammary tumor-derived cells transduced in vitro with empty vector virus (Neu/PERK loxP/loxP ) or Cre-expressing retrovirus (Neu/PERK Δ/Δ ) 28 days post-transplant (n=4). Representative image of tumors are provided. All p-values determined by Student t-test. (E) Western analysis of transgenic ErbB2 and PERK expression following infection of mouse mammary tumor-derived cells with control (Neu/PERK loxP/loxP ) or Cre-expressing retrovirus (Neu/PERK Δ/Δ ). Thapsigargin treatment (50nM, 1h) was used to demonstrate that PERK is functional.
Figure Legend Snippet: PERK expression is maintained in cancer cells wherein it regulates tumor expansion in vivo (A) PERK protein levels were measured by immunoprecipitation (IP) followed by Western blot analysis in the following cell lines: MCF10A (1), MCF7 (2), T47D (3), MDA-MB231 (4), MDA-MB468 (5), TE3 (6), TE7 (7), KYSE 520 (8). (B) PERK protein levels following shRNA targeting of PERK. (C) Parental MDA-MB468 cell line, shPERK-transduced cells (shPERK), and shPERK-transduced cells reconstituted with mouse Myc-PERK (+mPERK) were treated with 2μg/ml tunicamycin for the indicated intervals. Western analysis for ATF4, CHOP, or β-actin. (D) Volume of orthotopic tumors formed from the mouse mammary tumor-derived cells transduced in vitro with empty vector virus (Neu/PERK loxP/loxP ) or Cre-expressing retrovirus (Neu/PERK Δ/Δ ) 28 days post-transplant (n=4). Representative image of tumors are provided. All p-values determined by Student t-test. (E) Western analysis of transgenic ErbB2 and PERK expression following infection of mouse mammary tumor-derived cells with control (Neu/PERK loxP/loxP ) or Cre-expressing retrovirus (Neu/PERK Δ/Δ ). Thapsigargin treatment (50nM, 1h) was used to demonstrate that PERK is functional.

Techniques Used: Expressing, In Vivo, Immunoprecipitation, Western Blot, Multiple Displacement Amplification, shRNA, Derivative Assay, In Vitro, Plasmid Preparation, Transgenic Assay, Infection, Functional Assay

14) Product Images from "Loss of periostin/OSF-2 in ErbB2/Neu-driven tumors results in androgen receptor-positive molecular apocrine-like tumors with reduced Notch1 activity"

Article Title: Loss of periostin/OSF-2 in ErbB2/Neu-driven tumors results in androgen receptor-positive molecular apocrine-like tumors with reduced Notch1 activity

Journal: Breast Cancer Research : BCR

doi: 10.1186/s13058-014-0513-8

Loss of Postn does not affect tumor growth in MMTV-Neu mice. (A and B) Representative mammary gland whole mounts from 4 month old virgin females showing multiple tumor foci in both wildtype and Postn(-/-) mice. (C-F) H E staining of representative tumors collected at end point from wildtype (C and E) and Postn-null (D and F) mice. Both cystic (C and D) and solid tumors (E and F) were observed. (G) Kaplan-Meier survival analysis of tumor-bearing mice. Log rank testing showed no significant delay in tumor progression for Postn(-/-) mice when compared to heterozygotes or wildtype mice. All three genotypes reached end point with similar kinetics. (H-K) Paraffin-embedded sections were stained for ErbB2 (H and J) or Postn (I and K) . Tumors arising in Postn-null females retained Neu expression. In Neu-induced tumors, Postn expression was restricted to the stromal compartment and was never found in the tumor cells. (L) Western blot analysis of mammary tumors (T) and whole gland (G) lysates. Supporting the IHC data, Postn was found to be expressed at low levels in tumors containing a small amount of stromal fibroblasts.
Figure Legend Snippet: Loss of Postn does not affect tumor growth in MMTV-Neu mice. (A and B) Representative mammary gland whole mounts from 4 month old virgin females showing multiple tumor foci in both wildtype and Postn(-/-) mice. (C-F) H E staining of representative tumors collected at end point from wildtype (C and E) and Postn-null (D and F) mice. Both cystic (C and D) and solid tumors (E and F) were observed. (G) Kaplan-Meier survival analysis of tumor-bearing mice. Log rank testing showed no significant delay in tumor progression for Postn(-/-) mice when compared to heterozygotes or wildtype mice. All three genotypes reached end point with similar kinetics. (H-K) Paraffin-embedded sections were stained for ErbB2 (H and J) or Postn (I and K) . Tumors arising in Postn-null females retained Neu expression. In Neu-induced tumors, Postn expression was restricted to the stromal compartment and was never found in the tumor cells. (L) Western blot analysis of mammary tumors (T) and whole gland (G) lysates. Supporting the IHC data, Postn was found to be expressed at low levels in tumors containing a small amount of stromal fibroblasts.

Techniques Used: Mouse Assay, Staining, Expressing, Western Blot, Immunohistochemistry

15) Product Images from "ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells"

Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-08-0316

ErbB2, Src, and STAT3 form a complex in ErbB2 overexpressing cells. Whole cell lysates from A. 435.ErbB2 or B. SKBR3 cells were immunoprecipitated (IP) with the indicated antibodies followed by immunoblot analysis for ErbB2, STAT3, and Src. Input lanes represent 10% of total protein immunoprecipitated. Asterisk indicates IgG heavy chain.
Figure Legend Snippet: ErbB2, Src, and STAT3 form a complex in ErbB2 overexpressing cells. Whole cell lysates from A. 435.ErbB2 or B. SKBR3 cells were immunoprecipitated (IP) with the indicated antibodies followed by immunoblot analysis for ErbB2, STAT3, and Src. Input lanes represent 10% of total protein immunoprecipitated. Asterisk indicates IgG heavy chain.

Techniques Used: Immunoprecipitation

p21 Cip1 transcriptional activation by ErbB2 is dependant on STAT3 protein. A. 435.ErbB2 and 231.ErbB2 cells treated with 300 nM of Mismatch (MM) or STAT3 Antisense (AS) oligonucleotides. Cells were harvested 5 days post-transfection and analyzed by Western blot with the indicated antibodies. B. Standardized firefly luciferase activity of the wild type p21 promoter (pGL3-p21−2400) reporter plasmid in 435.ErbB2 and 231.ErbB2 cells treated with either STAT3 MM or AS oligonucleotides. Fold reduction was determined by standardizing to Mismatch control treated sample for each cell line. Error bars represent S.D.
Figure Legend Snippet: p21 Cip1 transcriptional activation by ErbB2 is dependant on STAT3 protein. A. 435.ErbB2 and 231.ErbB2 cells treated with 300 nM of Mismatch (MM) or STAT3 Antisense (AS) oligonucleotides. Cells were harvested 5 days post-transfection and analyzed by Western blot with the indicated antibodies. B. Standardized firefly luciferase activity of the wild type p21 promoter (pGL3-p21−2400) reporter plasmid in 435.ErbB2 and 231.ErbB2 cells treated with either STAT3 MM or AS oligonucleotides. Fold reduction was determined by standardizing to Mismatch control treated sample for each cell line. Error bars represent S.D.

Techniques Used: Activation Assay, Transfection, Western Blot, Luciferase, Activity Assay, Plasmid Preparation

Inhibition of STAT3 sensitized ErbB2 overexpressing breast cancer cells to Taxol. A. 435.ErbB2 and SKBR3 cells were treated with 50 μM of STAT3 inhibitory peptide or control peptide for 24 hours. Nuclear extracts were then subjected to EMSA. Band shifts indicated a STAT3 protein:DNA complex. Oct-1 protein:DNA binding serves as an loading control. B. 435.ErbB2 and SKBR3 cells were treated with control (CP) or STAT3 inhibitor peptide (SP) (100 μM for 435.ErbB2 and 50 μM for SKBR3) for 12 hours. Cells were then treated with different concentrations of Taxol for 24 hours. Cell growth inhibition was determined by MTS assay. Error bars represent S.D. Inset: Representative Western blot for p21 Cip1 and β-actin (loading control) from cells treated with either control (CP) or STAT3 inhibitor peptide (SP) treatment (** p = 0.004; * p = 0.016; t-test).
Figure Legend Snippet: Inhibition of STAT3 sensitized ErbB2 overexpressing breast cancer cells to Taxol. A. 435.ErbB2 and SKBR3 cells were treated with 50 μM of STAT3 inhibitory peptide or control peptide for 24 hours. Nuclear extracts were then subjected to EMSA. Band shifts indicated a STAT3 protein:DNA complex. Oct-1 protein:DNA binding serves as an loading control. B. 435.ErbB2 and SKBR3 cells were treated with control (CP) or STAT3 inhibitor peptide (SP) (100 μM for 435.ErbB2 and 50 μM for SKBR3) for 12 hours. Cells were then treated with different concentrations of Taxol for 24 hours. Cell growth inhibition was determined by MTS assay. Error bars represent S.D. Inset: Representative Western blot for p21 Cip1 and β-actin (loading control) from cells treated with either control (CP) or STAT3 inhibitor peptide (SP) treatment (** p = 0.004; * p = 0.016; t-test).

Techniques Used: Inhibition, Binding Assay, MTS Assay, Western Blot

Src is required for ErbB2 mediated STAT3 activation and p21 Cip1 upregulation. A. ErbB2 overexpressing cells were treated with 5 μM control (PP3) or Src inhibitor (PP2). Cell lysates were collected and analyzed by Western blotting. B. 435.ErbB2 and SKBR3 cells were transfected with the p21 Cip1 reporter plasmids wild type (pGL3-p21−2400) or SIE mutant (pGL3-p21−2400mSIE) and treated with 5 μM PP3 or PP2 for 24 hours. Cell lysates were collected and luciferase activities were measured and standardized by transfection efficiency using renilla values. Error bars represent S.D. C. Indicated cells were transfected with either vector control (Vec) or dominant negative Src mutant (pSRC-DN) for 24 hours. Lysates were analyzed by Western blotting. D. Cells were co-transfected with vector control or pSrc-DN plasmids and the pGL3-p21−2400 reporter plasmid. Luciferase activities were measured as in (C).
Figure Legend Snippet: Src is required for ErbB2 mediated STAT3 activation and p21 Cip1 upregulation. A. ErbB2 overexpressing cells were treated with 5 μM control (PP3) or Src inhibitor (PP2). Cell lysates were collected and analyzed by Western blotting. B. 435.ErbB2 and SKBR3 cells were transfected with the p21 Cip1 reporter plasmids wild type (pGL3-p21−2400) or SIE mutant (pGL3-p21−2400mSIE) and treated with 5 μM PP3 or PP2 for 24 hours. Cell lysates were collected and luciferase activities were measured and standardized by transfection efficiency using renilla values. Error bars represent S.D. C. Indicated cells were transfected with either vector control (Vec) or dominant negative Src mutant (pSRC-DN) for 24 hours. Lysates were analyzed by Western blotting. D. Cells were co-transfected with vector control or pSrc-DN plasmids and the pGL3-p21−2400 reporter plasmid. Luciferase activities were measured as in (C).

Techniques Used: Activation Assay, Western Blot, Transfection, Mutagenesis, Luciferase, Plasmid Preparation, Dominant Negative Mutation

Inhibition of Src kinase sensitized ErbB2 overexpressing breast cancer cells to Taxol. A. 435.ErbB2 cells were treated as indicated for 18 hours. Cell lysates were collected and analyzed by western blot analysis for the indicated antibodies. Numbers represent relative intensity of each band compared to the non-treated (NT) control. Total STAT3 was used as the loading control for both P-STAT3-Y705 and p21. B. 435.ErbB2 cells were treated with DMSO (control) or AZD0530 for 18 hours. Chromatin Immunoprecipitation (ChIP assay) for SIE p21 Cip1 promoter sequence was performed using antibodies against histone and STAT3. IgG and Histone immunoprecipitation served as negative and positive controls, respectively. Input represents 10% of the total. C. 435.ErbB2 and SKBR3 cells were treated with DMSO only or AZD0530 (2 μM) for 12 hours. After 12 hours, cell media was replaced with medium containing either Taxol (50 nM) only, AZD0530 only or combination of Taxol and AZD0530. Cell growth inhibition was determined by MTS assay at 36 hours. Results were normalized to DMSO controls (** p = 0.002; * p = 0.013; t-test).
Figure Legend Snippet: Inhibition of Src kinase sensitized ErbB2 overexpressing breast cancer cells to Taxol. A. 435.ErbB2 cells were treated as indicated for 18 hours. Cell lysates were collected and analyzed by western blot analysis for the indicated antibodies. Numbers represent relative intensity of each band compared to the non-treated (NT) control. Total STAT3 was used as the loading control for both P-STAT3-Y705 and p21. B. 435.ErbB2 cells were treated with DMSO (control) or AZD0530 for 18 hours. Chromatin Immunoprecipitation (ChIP assay) for SIE p21 Cip1 promoter sequence was performed using antibodies against histone and STAT3. IgG and Histone immunoprecipitation served as negative and positive controls, respectively. Input represents 10% of the total. C. 435.ErbB2 and SKBR3 cells were treated with DMSO only or AZD0530 (2 μM) for 12 hours. After 12 hours, cell media was replaced with medium containing either Taxol (50 nM) only, AZD0530 only or combination of Taxol and AZD0530. Cell growth inhibition was determined by MTS assay at 36 hours. Results were normalized to DMSO controls (** p = 0.002; * p = 0.013; t-test).

Techniques Used: Inhibition, Western Blot, Chromatin Immunoprecipitation, Sequencing, Immunoprecipitation, MTS Assay

Constitutive activation of STAT3 in ErbB2 overexpressing cells results in p21 Cip1 transcriptional upregulation. A. left , schematic diagram of the 5’ promoter region of the p21 gene. White boxes represent STAT SIE location on promoter; black box represents mutated SIE. right , Luciferase activity of extracts prepared from 435.Vec and 435.ErbB2 cells transfected with the indicated p21 Cip1 promoter reporter constructs. Bars indicate luciferase activity standardized to vector control for each construct. Error bars represent standard deviation (S.D.) B. Lysates from SKBR3, along with MDA-MB--435 and MDA-MB-231 breast cancer cells stably transfected with either control vector or wild-type ErbB2 were analyzed by Western blotting with the indicated antibodies. C. Nuclear extracts were collected from ErbB2 stable transfectants and vector control cells and SKBR3 cells for Electrophoretic Mobility Shift Assay (EMSA) analysis. Arrow indicates STAT protein:DNA complexes. Asterisk indicates addition of non-radiolabeled competitor probe. D. Chromatin Immunoprecipitation (ChIP assay) for SIE p21 Cip1 promoter sequence using antibodies against histone and STAT3 in ErbB2 overexpressing cells. Histone and IgG immunoprecipitation served as positive and negative controls, respectively. Input represents 10% of the total.
Figure Legend Snippet: Constitutive activation of STAT3 in ErbB2 overexpressing cells results in p21 Cip1 transcriptional upregulation. A. left , schematic diagram of the 5’ promoter region of the p21 gene. White boxes represent STAT SIE location on promoter; black box represents mutated SIE. right , Luciferase activity of extracts prepared from 435.Vec and 435.ErbB2 cells transfected with the indicated p21 Cip1 promoter reporter constructs. Bars indicate luciferase activity standardized to vector control for each construct. Error bars represent standard deviation (S.D.) B. Lysates from SKBR3, along with MDA-MB--435 and MDA-MB-231 breast cancer cells stably transfected with either control vector or wild-type ErbB2 were analyzed by Western blotting with the indicated antibodies. C. Nuclear extracts were collected from ErbB2 stable transfectants and vector control cells and SKBR3 cells for Electrophoretic Mobility Shift Assay (EMSA) analysis. Arrow indicates STAT protein:DNA complexes. Asterisk indicates addition of non-radiolabeled competitor probe. D. Chromatin Immunoprecipitation (ChIP assay) for SIE p21 Cip1 promoter sequence using antibodies against histone and STAT3 in ErbB2 overexpressing cells. Histone and IgG immunoprecipitation served as positive and negative controls, respectively. Input represents 10% of the total.

Techniques Used: Activation Assay, Luciferase, Activity Assay, Transfection, Construct, Plasmid Preparation, Standard Deviation, Multiple Displacement Amplification, Stable Transfection, Western Blot, Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, Sequencing, Immunoprecipitation

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Centrifugation:

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Article Title: Destabilization of ERBB2 transcripts by targeting 3? UTR mRNA associated HuR and histone deacetylase-6 (HDAC6)
Article Snippet: Following removal of nuclei by centrifugation, the cytoplasmic lysate was brought to 50 mM KCl and incubated at 4°C for 5 hours with either 0.2 μg of HuR, HDAC6 or control antibodies together with 12 μl of protein G Sepharose beads (GE Healthcare, Uppsala, Sweeden) per ml of lysate. .. Antibodies used included ERBB2 (Calbiochem, San Diego, CA), β-actin (Abcam, Cambridge, MA), α-tubulin (Sigma), acetylated α-tubulin (Sigma), AUF1 (Upstate), HuR (mouse 3A2, Santa Cruz Biotechnology), Lex A (mouse control Ab for HuR IPs, Santa Cruz Biotechnology), HDAC6 (sc-11420, Santa Cruz Biotechnology), ERα (rabbit control Ab for HDAC6 IPs, Santa Cruz Biotechnology) acetylated H4 (Upstate) or K12-acetylated H4 (Cell Signaling Technology, Danvers, MA ).

Positive Control:

Article Title: The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells
Article Snippet: Immunohistochemistry Five micron thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in TBST + 1% BSA w/v), cleaved Caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1000 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; cat#OP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v). .. For erbB2 and erbB3, SKBR3 cells were used as a positive control.

Article Title: Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer
Article Snippet: Immunohistochemistry (IHC) Five-micron-thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki-67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in Tris-buffered saline and Tween 20 (TBST) + 1% BSA w/v), cleaved caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1,000 in TBST + 1% BSA w/v), Survivin (Epitomics, Burlingame, CA, USA; rabbit monoclonal EP2880Y; cat# 2463; dilution 1:100 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; cat#OP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA, USA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v). .. For erbB2 and erbB3, SKBR3 cells were used as a positive control.

Cytotoxicity Assay:

Article Title: Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane
Article Snippet: Specific siRNA for the knockdown of ErbB2 was purchased from Sigma-Aldrich (MISSION® siRNA SIHK0723) and a plasmid vector containing wild-type Myc-DDK-tagged ErbB2 (cat no. RC212583) was purchased from OriGene Technologies, Inc., Rockville, MD, USA. .. Forty-eight hours after transfection, whole-cell lysates were prepared for further analysis by Western blot and cytotoxicity assay.

Incubation:

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: .. The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated. .. For the inverse immune precipitation, lysates of transfected or untransfected MCF7 cells were prepared as described above.

Article Title: Destabilization of ERBB2 transcripts by targeting 3? UTR mRNA associated HuR and histone deacetylase-6 (HDAC6)
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Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells
Article Snippet: .. Membranes were incubated with the following antibodies: ErbB2 (Calbiochem); STAT1, STAT3, phospho-STAT3-pY705, anti-p21Cip1 (Cell Signaling Technology, Danvers, MA), anti-Src (Santa Cruz Technology, Santa Cruz, CA), β-actin (Sigma, St. Louis, MO). .. Secondary antibody detection utilized Alexa-Fluor secondary antibodies (Invitrogen Molecular Probes).

Activity Assay:

Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism
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Expressing:

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
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Western Blot:

Article Title: Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane
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Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism
Article Snippet: .. Antibodies and Reagents The antibodies were purchased from: ErbB2 (OP15, Calbiochem), mtHSP70 (MA3-028, Thermo Scientific), VDAC1 (sc-58649, Santa Cruz), Bcl-2 (Rabbit mAb #2870, Cell Signaling), Integrin β (610467, BD Bioscience), Prohibitin (EP2803Y, Abcam), KDEL (sc-33806, Santa Cruz), EEA1(sc-130017, Santa Cruz), Golgi Complex (NB37-10UG, Calbiochem), LAMP2 (sc-18822, Santa Cruz), Lamin B1 (MA1-06103, Thermo Scientific), Mitochondrial Complex I subunit NDUFB8, Complex II subunit 30kDa, Complex III subunit Core 2, Complex IV subunit II, and ATP synthase subunit alpha antibodies mix were MitoProfile® Total OXPHOS Human WB Antibody Cocktail (MS601, Mitoscience). siRNAs for mtHSP70(1) (SASI_Hs01_00216924) and mtHSP70(2) (MISSION® siRNA SASI_Hs01_00216923) were purchased from Sigma. .. Protease K was purchased from Sigma; the immunoprecipitation assay was conducted using Pierce Co-immunoprecipitation Kit (Cat: 26149) from Thermo Scientific.

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: .. The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated. .. For the inverse immune precipitation, lysates of transfected or untransfected MCF7 cells were prepared as described above.

Article Title: The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells
Article Snippet: .. Antibodies used for western blots were as follows: erbB2 (EMD Chemicals, Inc., Gibbstown, NJ); erbB3 and P-erbB2 (Tyr1248) (LabVision Corp., Fremont, CA); P-erbB3 (Tyr1289), P-MAPK (Thr202/Tyr204), MAPK, P-Akt (Ser473), and Akt (Cell Signaling Technology, Inc., Beverly, MA); Cyclin D1 (M-20), E2F1 (KH95), and p27kip1 (F-8) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and β-actin (Sigma Co., St. Louis, MO). .. Cells and cell culture Human breast cancer cell lines SKBR3 and BT474 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

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Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage
Article Snippet: .. Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore). .. Lentivirus, Retrovirus shRNA/siRNA 293T cells were transfected with PMDL, VSVG, REV and pLKO.1 containing shRNA against PERK (IDTRCN0000001401, Open Biosystem) or pLKO.1 empty vector as control using Lipofectamine Plus (Invitrogene) for stable knockdown or FuGene (Roche) for acute knockdown experiment.

Article Title: A novel luciferase based reporter system to monitor activation of the ErbB2/Her2/neu pathway non-invasively during radiotherapy
Article Snippet: Paragraph title: Cell culture and western blotting ... The following primary antibodies were used: ErbB2 (Calbiochem, San Diego, California) 1:1000, pN2A (phosphor-ErbB2, Neomarkers, Fremont, California) 1:1000, Shc, Phospho-Shc, mitogen-activated protein kinase (MAPK) and P-MAPK (Cell Signaling Technology, Beverly, Massachusetts) 1:1000, Beta Actin (Lab Vision Corp, Fremont, CA) 1:1000.

Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells
Article Snippet: Paragraph title: Western Blot and Immunoprecipitation Analysis ... Membranes were incubated with the following antibodies: ErbB2 (Calbiochem); STAT1, STAT3, phospho-STAT3-pY705, anti-p21Cip1 (Cell Signaling Technology, Danvers, MA), anti-Src (Santa Cruz Technology, Santa Cruz, CA), β-actin (Sigma, St. Louis, MO).

Article Title: Increased erbB3 promotes erbB2/neu-driven mammary tumor proliferation and co-targeting of erbB2/erbB3 receptors exhibits potent inhibitory effects on breast cancer cells
Article Snippet: .. Antibodies used for Western blots were from the following sources: erbB2 (c-neu Ab-3, EMD Chemicals, Inc., Gibbstown, NJ); phospho-erbB2 (P-erbB2, clone PN2A), and erbB3 (Ab-7) (Lab Vision/NeoMarkers, Inc., Fremont, CA); phospho-erbB3 (P-erbB3, clone 21D3), phospho-Akt (Ser473), Akt, P-MAPK (Thr202/Tyr204), MAPK (Cell Signaling Technology, Inc., Danvers, MA); and β-actin (clone AC-74, Sigma-Aldrich Co., St. Louis, MO). .. Murine mammary tumor cell lines 85815 and 85819 were established from mammary tumors derived from the wild type (wt) rat c- erbB2/neu transgenic mice [ , ].

Article Title: Loss of periostin/OSF-2 in ErbB2/Neu-driven tumors results in androgen receptor-positive molecular apocrine-like tumors with reduced Notch1 activity
Article Snippet: Paragraph title: Western blot analysis and antibodies ... The following antibodies were purchased and used according to the manufacturer’s protocols: Postn (R & D Systems, Minneapolis, MN, USA), pAkt-S473, Akt, NICD, Notch1 (Cell Signaling, Danvers, MA, USA), cyclin D1, (Santa Cruz Technology, Dallas, TX, USA), pY397FAK, focal adhesion kinase (FAK) (BD Biosciences, San Jose, CA, USA), androgen receptor, CD31 (Abcam, Cambridge, UK), ErbB2 (Calbiochem, clone ab-3), Ki67, non-phospho beta catenin (EMD Millipore, Billerica, MA, USA), tubulin (Sigma-Aldrich, St Louis, MO, USA), γ-secretase inhibitor, (In solution inhibitor X; Calbiochem).

Over Expression:

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism
Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com . .. The siRNA oligonucleotides for ErbB2 was purchased from Sigma-Aldrich, with a scrambled siRNA (Sigma-Aldrich) used as a control.

Hybridization:

Article Title: Destabilization of ERBB2 transcripts by targeting 3? UTR mRNA associated HuR and histone deacetylase-6 (HDAC6)
Article Snippet: Equal aliquots of immunoprecipitated material were electrophoresed using 4–12% gradient gels (Invitrogen), transferred to nitrocellulose (Amersham Bioscience) and probed for HuR and HDAC6 in hybridization buffers of 20 mM Tris pH7.5, 130 mM NaCl, 0.05% Tween-20 with 5% non-fat milk. .. Antibodies used included ERBB2 (Calbiochem, San Diego, CA), β-actin (Abcam, Cambridge, MA), α-tubulin (Sigma), acetylated α-tubulin (Sigma), AUF1 (Upstate), HuR (mouse 3A2, Santa Cruz Biotechnology), Lex A (mouse control Ab for HuR IPs, Santa Cruz Biotechnology), HDAC6 (sc-11420, Santa Cruz Biotechnology), ERα (rabbit control Ab for HDAC6 IPs, Santa Cruz Biotechnology) acetylated H4 (Upstate) or K12-acetylated H4 (Cell Signaling Technology, Danvers, MA ).

Immunohistochemistry:

Article Title: The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells
Article Snippet: .. Immunohistochemistry Five micron thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in TBST + 1% BSA w/v), cleaved Caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1000 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; cat#OP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v). .. For erbB2 and erbB3, SKBR3 cells were used as a positive control.

Article Title: Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer
Article Snippet: .. Immunohistochemistry (IHC) Five-micron-thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki-67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in Tris-buffered saline and Tween 20 (TBST) + 1% BSA w/v), cleaved caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1,000 in TBST + 1% BSA w/v), Survivin (Epitomics, Burlingame, CA, USA; rabbit monoclonal EP2880Y; cat# 2463; dilution 1:100 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; cat#OP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA, USA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v). .. For erbB2 and erbB3, SKBR3 cells were used as a positive control.

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage
Article Snippet: .. Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore). .. Lentivirus, Retrovirus shRNA/siRNA 293T cells were transfected with PMDL, VSVG, REV and pLKO.1 containing shRNA against PERK (IDTRCN0000001401, Open Biosystem) or pLKO.1 empty vector as control using Lipofectamine Plus (Invitrogene) for stable knockdown or FuGene (Roche) for acute knockdown experiment.

Stable Transfection:

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated. .. Endogenous ERBB3 (C17, Santa Cruz) in untransfected MCF7 cells or Dendra2 fusion proteins in stably transfected MCF7 cells were detected by Western blotting after binding and processing at room temperature as indicated above.

FLAG-tag:

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: Equal numbers of MCF7 cells were seeded and transfected with pFlag-ERBB2 or pFlag-ERBB3, a constitutive expression vector carrying an N-terminal FLAG epitope tag. .. The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated.

Protease Inhibitor:

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: Cells were washed in PBS supplemented with 1 mM phenylmethylsulfonyl fluoride and lysed in TMISV buffer (150 mM Tris–HCl, pH 7.5, 150 mM NaCl, 20 mM NaMoO4 , 0.09% Igepal, 1 mM NaVO4 , 1 μg/ml leupeptin, 1 μg/ml aprotinin, and protease inhibitor cocktail (Sigma)). .. The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated.

Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells
Article Snippet: Total cell lysates were collected in IP lysis buffer (20 mM Tris pH 7.5, 150 mM, NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM sodium orthovanadate, protease inhibitor cocktail, 1 mM PMSF). .. Membranes were incubated with the following antibodies: ErbB2 (Calbiochem); STAT1, STAT3, phospho-STAT3-pY705, anti-p21Cip1 (Cell Signaling Technology, Danvers, MA), anti-Src (Santa Cruz Technology, Santa Cruz, CA), β-actin (Sigma, St. Louis, MO).

Cell Culture:

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: Cells were scraped of the cell culture dishes, and lysates were passed repeatedly through a 26-gauge needle, followed by centrifugation at 15,000× g for 15 min. .. The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated.

Article Title: A novel luciferase based reporter system to monitor activation of the ErbB2/Her2/neu pathway non-invasively during radiotherapy
Article Snippet: Paragraph title: Cell culture and western blotting ... The following primary antibodies were used: ErbB2 (Calbiochem, San Diego, California) 1:1000, pN2A (phosphor-ErbB2, Neomarkers, Fremont, California) 1:1000, Shc, Phospho-Shc, mitogen-activated protein kinase (MAPK) and P-MAPK (Cell Signaling Technology, Beverly, Massachusetts) 1:1000, Beta Actin (Lab Vision Corp, Fremont, CA) 1:1000.

Binding Assay:

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated. .. Endogenous ERBB3 (C17, Santa Cruz) in untransfected MCF7 cells or Dendra2 fusion proteins in stably transfected MCF7 cells were detected by Western blotting after binding and processing at room temperature as indicated above.

Lactate Assay:

Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism
Article Snippet: Antibodies and Reagents The antibodies were purchased from: ErbB2 (OP15, Calbiochem), mtHSP70 (MA3-028, Thermo Scientific), VDAC1 (sc-58649, Santa Cruz), Bcl-2 (Rabbit mAb #2870, Cell Signaling), Integrin β (610467, BD Bioscience), Prohibitin (EP2803Y, Abcam), KDEL (sc-33806, Santa Cruz), EEA1(sc-130017, Santa Cruz), Golgi Complex (NB37-10UG, Calbiochem), LAMP2 (sc-18822, Santa Cruz), Lamin B1 (MA1-06103, Thermo Scientific), Mitochondrial Complex I subunit NDUFB8, Complex II subunit 30kDa, Complex III subunit Core 2, Complex IV subunit II, and ATP synthase subunit alpha antibodies mix were MitoProfile® Total OXPHOS Human WB Antibody Cocktail (MS601, Mitoscience). siRNAs for mtHSP70(1) (SASI_Hs01_00216924) and mtHSP70(2) (MISSION® siRNA SASI_Hs01_00216923) were purchased from Sigma. .. Mitochondrial membrane potential kit was purchased from BD™ MitoScreen (JC-1); Glucose uptake was measured using the Amplex Red Glucose/Glucose Oxidase Assay Kit (Molecular Probes, Carlsbad, CA, USA); Lactate production in the medium was detected by using the Lactate Assay Kit (BioVision, Mountain View, CA, USA).

Immunofluorescence:

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage
Article Snippet: .. Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore). .. Lentivirus, Retrovirus shRNA/siRNA 293T cells were transfected with PMDL, VSVG, REV and pLKO.1 containing shRNA against PERK (IDTRCN0000001401, Open Biosystem) or pLKO.1 empty vector as control using Lipofectamine Plus (Invitrogene) for stable knockdown or FuGene (Roche) for acute knockdown experiment.

Multiple Displacement Amplification:

Article Title: A novel luciferase based reporter system to monitor activation of the ErbB2/Her2/neu pathway non-invasively during radiotherapy
Article Snippet: 293T, MDA-MB231, and BT474 cell lines were maintained in DMEM supplemented with 10% FBS. .. The following primary antibodies were used: ErbB2 (Calbiochem, San Diego, California) 1:1000, pN2A (phosphor-ErbB2, Neomarkers, Fremont, California) 1:1000, Shc, Phospho-Shc, mitogen-activated protein kinase (MAPK) and P-MAPK (Cell Signaling Technology, Beverly, Massachusetts) 1:1000, Beta Actin (Lab Vision Corp, Fremont, CA) 1:1000.

Isolation:

Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism
Article Snippet: Antibodies and Reagents The antibodies were purchased from: ErbB2 (OP15, Calbiochem), mtHSP70 (MA3-028, Thermo Scientific), VDAC1 (sc-58649, Santa Cruz), Bcl-2 (Rabbit mAb #2870, Cell Signaling), Integrin β (610467, BD Bioscience), Prohibitin (EP2803Y, Abcam), KDEL (sc-33806, Santa Cruz), EEA1(sc-130017, Santa Cruz), Golgi Complex (NB37-10UG, Calbiochem), LAMP2 (sc-18822, Santa Cruz), Lamin B1 (MA1-06103, Thermo Scientific), Mitochondrial Complex I subunit NDUFB8, Complex II subunit 30kDa, Complex III subunit Core 2, Complex IV subunit II, and ATP synthase subunit alpha antibodies mix were MitoProfile® Total OXPHOS Human WB Antibody Cocktail (MS601, Mitoscience). siRNAs for mtHSP70(1) (SASI_Hs01_00216924) and mtHSP70(2) (MISSION® siRNA SASI_Hs01_00216923) were purchased from Sigma. .. Mitotracker was purchased from Invitrogen; Intact mitochondria were prepared and purified using Qproteome Mitochondria Isolation Kit from Qiagen; ADP/ATP assay kit was purchased from BioVison; Oxygen Consumption assay kit was from BD Bioscience and Mitochondrial Complex activity assay kits (I, II, IV and V) were purchased from Mitoscience.

Transfection:

Article Title: Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane
Article Snippet: Paragraph title: Small interfering (si)RNA and plasmid DNA transfection ... Specific siRNA for the knockdown of ErbB2 was purchased from Sigma-Aldrich (MISSION® siRNA SIHK0723) and a plasmid vector containing wild-type Myc-DDK-tagged ErbB2 (cat no. RC212583) was purchased from OriGene Technologies, Inc., Rockville, MD, USA.

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: Equal numbers of MCF7 cells were seeded and transfected with pFlag-ERBB2 or pFlag-ERBB3, a constitutive expression vector carrying an N-terminal FLAG epitope tag. .. The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated.

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism
Article Snippet: Paragraph title: Plasmid DNA and siRNA transfections ... The siRNA oligonucleotides for ErbB2 was purchased from Sigma-Aldrich, with a scrambled siRNA (Sigma-Aldrich) used as a control.

Labeling:

Article Title: Increased erbB3 promotes erbB2/neu-driven mammary tumor proliferation and co-targeting of erbB2/erbB3 receptors exhibits potent inhibitory effects on breast cancer cells
Article Snippet: The lentiviral vector pLKO.1-puro containing a mouse specific erbB3 shRNA (pLKO.1-ErbB3shRNA: TRCN0000023429~33, labeled as sh-1~5 in text) was from Functional Genomics Facility at the University of Colorado at Boulder. .. Antibodies used for Western blots were from the following sources: erbB2 (c-neu Ab-3, EMD Chemicals, Inc., Gibbstown, NJ); phospho-erbB2 (P-erbB2, clone PN2A), and erbB3 (Ab-7) (Lab Vision/NeoMarkers, Inc., Fremont, CA); phospho-erbB3 (P-erbB3, clone 21D3), phospho-Akt (Ser473), Akt, P-MAPK (Thr202/Tyr204), MAPK (Cell Signaling Technology, Inc., Danvers, MA); and β-actin (clone AC-74, Sigma-Aldrich Co., St. Louis, MO).

Purification:

Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism
Article Snippet: Antibodies and Reagents The antibodies were purchased from: ErbB2 (OP15, Calbiochem), mtHSP70 (MA3-028, Thermo Scientific), VDAC1 (sc-58649, Santa Cruz), Bcl-2 (Rabbit mAb #2870, Cell Signaling), Integrin β (610467, BD Bioscience), Prohibitin (EP2803Y, Abcam), KDEL (sc-33806, Santa Cruz), EEA1(sc-130017, Santa Cruz), Golgi Complex (NB37-10UG, Calbiochem), LAMP2 (sc-18822, Santa Cruz), Lamin B1 (MA1-06103, Thermo Scientific), Mitochondrial Complex I subunit NDUFB8, Complex II subunit 30kDa, Complex III subunit Core 2, Complex IV subunit II, and ATP synthase subunit alpha antibodies mix were MitoProfile® Total OXPHOS Human WB Antibody Cocktail (MS601, Mitoscience). siRNAs for mtHSP70(1) (SASI_Hs01_00216924) and mtHSP70(2) (MISSION® siRNA SASI_Hs01_00216923) were purchased from Sigma. .. Mitotracker was purchased from Invitrogen; Intact mitochondria were prepared and purified using Qproteome Mitochondria Isolation Kit from Qiagen; ADP/ATP assay kit was purchased from BioVison; Oxygen Consumption assay kit was from BD Bioscience and Mitochondrial Complex activity assay kits (I, II, IV and V) were purchased from Mitoscience.

Staining:

Article Title: The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells
Article Snippet: .. Immunohistochemistry Five micron thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in TBST + 1% BSA w/v), cleaved Caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1000 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; cat#OP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v). .. For erbB2 and erbB3, SKBR3 cells were used as a positive control.

Article Title: Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer
Article Snippet: .. Immunohistochemistry (IHC) Five-micron-thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki-67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in Tris-buffered saline and Tween 20 (TBST) + 1% BSA w/v), cleaved caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1,000 in TBST + 1% BSA w/v), Survivin (Epitomics, Burlingame, CA, USA; rabbit monoclonal EP2880Y; cat# 2463; dilution 1:100 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; cat#OP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA, USA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v). .. For erbB2 and erbB3, SKBR3 cells were used as a positive control.

Chloramphenicol Acetyltransferase Assay:

Article Title: The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells
Article Snippet: .. Immunohistochemistry Five micron thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in TBST + 1% BSA w/v), cleaved Caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1000 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; catOP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v). .. For erbB2 and erbB3, SKBR3 cells were used as a positive control.

Article Title: Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer
Article Snippet: .. Immunohistochemistry (IHC) Five-micron-thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki-67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in Tris-buffered saline and Tween 20 (TBST) + 1% BSA w/v), cleaved caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1,000 in TBST + 1% BSA w/v), Survivin (Epitomics, Burlingame, CA, USA; rabbit monoclonal EP2880Y; cat# 2463; dilution 1:100 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; catOP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA, USA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v). .. For erbB2 and erbB3, SKBR3 cells were used as a positive control.

SDS Page:

Article Title: Mitochondrially targeted vitamin E succinate efficiently kills breast tumour-initiating cells in a complex II-dependent manner
Article Snippet: .. Western blotting (WB) Cells and homogenised tumour tissue were lysed, and total protein (30 μg) resolved by SDS-PAGE and transferred to PVDF membranes, which were probed with following antibodies: EpCAM, erbB2 (both from Sigma-Aldrich), caspase-9, caspase-8, cleaved caspase-3, VDAC, COX IV, SDHA (all from Cell Signaling), CD44, HSP60, actin (all from Abcam), CD133, PARP-1/2 (both from Santa Cruz), and SDHC (Novus Biologicals). .. ECL western blotting substrate (Thermo Scientific) and ChemiDoc™ XRS+ System (BioRad) were used to visualise and evaluate the blots.

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: .. The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated. .. For the inverse immune precipitation, lysates of transfected or untransfected MCF7 cells were prepared as described above.

Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells
Article Snippet: Lysates were subjected to SDS-PAGE followed by transfer to nitrocellulose membranes. .. Membranes were incubated with the following antibodies: ErbB2 (Calbiochem); STAT1, STAT3, phospho-STAT3-pY705, anti-p21Cip1 (Cell Signaling Technology, Danvers, MA), anti-Src (Santa Cruz Technology, Santa Cruz, CA), β-actin (Sigma, St. Louis, MO).

Article Title: Loss of periostin/OSF-2 in ErbB2/Neu-driven tumors results in androgen receptor-positive molecular apocrine-like tumors with reduced Notch1 activity
Article Snippet: Samples containing 40 μg of total protein were resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (PerkinElmer, Waltham, MA, USA). .. The following antibodies were purchased and used according to the manufacturer’s protocols: Postn (R & D Systems, Minneapolis, MN, USA), pAkt-S473, Akt, NICD, Notch1 (Cell Signaling, Danvers, MA, USA), cyclin D1, (Santa Cruz Technology, Dallas, TX, USA), pY397FAK, focal adhesion kinase (FAK) (BD Biosciences, San Jose, CA, USA), androgen receptor, CD31 (Abcam, Cambridge, UK), ErbB2 (Calbiochem, clone ab-3), Ki67, non-phospho beta catenin (EMD Millipore, Billerica, MA, USA), tubulin (Sigma-Aldrich, St Louis, MO, USA), γ-secretase inhibitor, (In solution inhibitor X; Calbiochem).

Plasmid Preparation:

Article Title: Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane
Article Snippet: .. Specific siRNA for the knockdown of ErbB2 was purchased from Sigma-Aldrich (MISSION® siRNA SIHK0723) and a plasmid vector containing wild-type Myc-DDK-tagged ErbB2 (cat no. RC212583) was purchased from OriGene Technologies, Inc., Rockville, MD, USA. .. Transfection was performed using the Oligofectamine™ transfection reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions.

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: Equal numbers of MCF7 cells were seeded and transfected with pFlag-ERBB2 or pFlag-ERBB3, a constitutive expression vector carrying an N-terminal FLAG epitope tag. .. The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated.

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism
Article Snippet: Paragraph title: Plasmid DNA and siRNA transfections ... The siRNA oligonucleotides for ErbB2 was purchased from Sigma-Aldrich, with a scrambled siRNA (Sigma-Aldrich) used as a control.

Article Title: Increased erbB3 promotes erbB2/neu-driven mammary tumor proliferation and co-targeting of erbB2/erbB3 receptors exhibits potent inhibitory effects on breast cancer cells
Article Snippet: The lentiviral vector pLKO.1-puro containing a mouse specific erbB3 shRNA (pLKO.1-ErbB3shRNA: TRCN0000023429~33, labeled as sh-1~5 in text) was from Functional Genomics Facility at the University of Colorado at Boulder. .. Antibodies used for Western blots were from the following sources: erbB2 (c-neu Ab-3, EMD Chemicals, Inc., Gibbstown, NJ); phospho-erbB2 (P-erbB2, clone PN2A), and erbB3 (Ab-7) (Lab Vision/NeoMarkers, Inc., Fremont, CA); phospho-erbB3 (P-erbB3, clone 21D3), phospho-Akt (Ser473), Akt, P-MAPK (Thr202/Tyr204), MAPK (Cell Signaling Technology, Inc., Danvers, MA); and β-actin (clone AC-74, Sigma-Aldrich Co., St. Louis, MO).

Functional Assay:

Article Title: Increased erbB3 promotes erbB2/neu-driven mammary tumor proliferation and co-targeting of erbB2/erbB3 receptors exhibits potent inhibitory effects on breast cancer cells
Article Snippet: The lentiviral vector pLKO.1-puro containing a mouse specific erbB3 shRNA (pLKO.1-ErbB3shRNA: TRCN0000023429~33, labeled as sh-1~5 in text) was from Functional Genomics Facility at the University of Colorado at Boulder. .. Antibodies used for Western blots were from the following sources: erbB2 (c-neu Ab-3, EMD Chemicals, Inc., Gibbstown, NJ); phospho-erbB2 (P-erbB2, clone PN2A), and erbB3 (Ab-7) (Lab Vision/NeoMarkers, Inc., Fremont, CA); phospho-erbB3 (P-erbB3, clone 21D3), phospho-Akt (Ser473), Akt, P-MAPK (Thr202/Tyr204), MAPK (Cell Signaling Technology, Inc., Danvers, MA); and β-actin (clone AC-74, Sigma-Aldrich Co., St. Louis, MO).

shRNA:

Article Title: Increased erbB3 promotes erbB2/neu-driven mammary tumor proliferation and co-targeting of erbB2/erbB3 receptors exhibits potent inhibitory effects on breast cancer cells
Article Snippet: The lentiviral vector pLKO.1-puro containing a mouse specific erbB3 shRNA (pLKO.1-ErbB3shRNA: TRCN0000023429~33, labeled as sh-1~5 in text) was from Functional Genomics Facility at the University of Colorado at Boulder. .. Antibodies used for Western blots were from the following sources: erbB2 (c-neu Ab-3, EMD Chemicals, Inc., Gibbstown, NJ); phospho-erbB2 (P-erbB2, clone PN2A), and erbB3 (Ab-7) (Lab Vision/NeoMarkers, Inc., Fremont, CA); phospho-erbB3 (P-erbB3, clone 21D3), phospho-Akt (Ser473), Akt, P-MAPK (Thr202/Tyr204), MAPK (Cell Signaling Technology, Inc., Danvers, MA); and β-actin (clone AC-74, Sigma-Aldrich Co., St. Louis, MO).

Two Tailed Test:

Article Title: Loss of periostin/OSF-2 in ErbB2/Neu-driven tumors results in androgen receptor-positive molecular apocrine-like tumors with reduced Notch1 activity
Article Snippet: Statistical analysis was performed using a two-tailed unpaired Student’s t test. .. The following antibodies were purchased and used according to the manufacturer’s protocols: Postn (R & D Systems, Minneapolis, MN, USA), pAkt-S473, Akt, NICD, Notch1 (Cell Signaling, Danvers, MA, USA), cyclin D1, (Santa Cruz Technology, Dallas, TX, USA), pY397FAK, focal adhesion kinase (FAK) (BD Biosciences, San Jose, CA, USA), androgen receptor, CD31 (Abcam, Cambridge, UK), ErbB2 (Calbiochem, clone ab-3), Ki67, non-phospho beta catenin (EMD Millipore, Billerica, MA, USA), tubulin (Sigma-Aldrich, St Louis, MO, USA), γ-secretase inhibitor, (In solution inhibitor X; Calbiochem).

Immunoprecipitation:

Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism
Article Snippet: Antibodies and Reagents The antibodies were purchased from: ErbB2 (OP15, Calbiochem), mtHSP70 (MA3-028, Thermo Scientific), VDAC1 (sc-58649, Santa Cruz), Bcl-2 (Rabbit mAb #2870, Cell Signaling), Integrin β (610467, BD Bioscience), Prohibitin (EP2803Y, Abcam), KDEL (sc-33806, Santa Cruz), EEA1(sc-130017, Santa Cruz), Golgi Complex (NB37-10UG, Calbiochem), LAMP2 (sc-18822, Santa Cruz), Lamin B1 (MA1-06103, Thermo Scientific), Mitochondrial Complex I subunit NDUFB8, Complex II subunit 30kDa, Complex III subunit Core 2, Complex IV subunit II, and ATP synthase subunit alpha antibodies mix were MitoProfile® Total OXPHOS Human WB Antibody Cocktail (MS601, Mitoscience). siRNAs for mtHSP70(1) (SASI_Hs01_00216924) and mtHSP70(2) (MISSION® siRNA SASI_Hs01_00216923) were purchased from Sigma. .. Protease K was purchased from Sigma; the immunoprecipitation assay was conducted using Pierce Co-immunoprecipitation Kit (Cat: 26149) from Thermo Scientific.

Article Title: Geldanamycin selectively targets the nascent form of ERBB3 for degradation
Article Snippet: .. The cleared lysate was incubated for 2 h on a rocker with or without antibodies against the N terminus of either ERBB2 (Ab-5, EMD Biosciences) or ERBB3 (Ab-4, EMD Biosciences), followed by incubation with protein A/G beads (Santa Cruz Biotech, CA, USA) for an additional 2 h. Immunoprecipitated samples were analyzed by SDS-PAGE and Western blotting against HSP90 (SPA-835, Assay Designs), ERBB2 (C18, Santa Cruz), or ERBB3 (C17, Santa Cruz) as indicated. .. For the inverse immune precipitation, lysates of transfected or untransfected MCF7 cells were prepared as described above.

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage
Article Snippet: Paragraph title: Immunoprecipitation and immunobloting ... Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore).

Article Title: Destabilization of ERBB2 transcripts by targeting 3? UTR mRNA associated HuR and histone deacetylase-6 (HDAC6)
Article Snippet: Paragraph title: Immunoblotting and immunoprecipitation ... Antibodies used included ERBB2 (Calbiochem, San Diego, CA), β-actin (Abcam, Cambridge, MA), α-tubulin (Sigma), acetylated α-tubulin (Sigma), AUF1 (Upstate), HuR (mouse 3A2, Santa Cruz Biotechnology), Lex A (mouse control Ab for HuR IPs, Santa Cruz Biotechnology), HDAC6 (sc-11420, Santa Cruz Biotechnology), ERα (rabbit control Ab for HDAC6 IPs, Santa Cruz Biotechnology) acetylated H4 (Upstate) or K12-acetylated H4 (Cell Signaling Technology, Danvers, MA ).

Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells
Article Snippet: Paragraph title: Western Blot and Immunoprecipitation Analysis ... Membranes were incubated with the following antibodies: ErbB2 (Calbiochem); STAT1, STAT3, phospho-STAT3-pY705, anti-p21Cip1 (Cell Signaling Technology, Danvers, MA), anti-Src (Santa Cruz Technology, Santa Cruz, CA), β-actin (Sigma, St. Louis, MO).

Lysis:

Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells
Article Snippet: Total cell lysates were collected in IP lysis buffer (20 mM Tris pH 7.5, 150 mM, NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM sodium orthovanadate, protease inhibitor cocktail, 1 mM PMSF). .. Membranes were incubated with the following antibodies: ErbB2 (Calbiochem); STAT1, STAT3, phospho-STAT3-pY705, anti-p21Cip1 (Cell Signaling Technology, Danvers, MA), anti-Src (Santa Cruz Technology, Santa Cruz, CA), β-actin (Sigma, St. Louis, MO).

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  • 99
    Millipore erbb2
    <t>ErbB2,</t> Src, and STAT3 form a complex in ErbB2 overexpressing cells. Whole cell lysates from A. 435.ErbB2 or B. SKBR3 cells were immunoprecipitated (IP) with the indicated antibodies followed by immunoblot analysis for ErbB2, STAT3, and Src. Input lanes represent 10% of total protein immunoprecipitated. Asterisk indicates IgG heavy chain.
    Erbb2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/erbb2/product/Millipore
    Average 99 stars, based on 60 article reviews
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    erbb2 - by Bioz Stars, 2020-02
    99/100 stars
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    76
    Millipore erbb2 signaling inhibitor
    inhbaa GOF promotes CM proliferation independently of <t>ErbB2</t> signaling and competes with m stnb GOF. a Experimental setup of EdU treatment, followed by fixation. b , c Tg(myl7:nlsDsRedExpress) hearts of wild-type sibling and inhbaa OE larvae at 120 hpf; α-DsRed (red), EdU (green). White arrowheads point to proliferating CMs (EdU + /DsRed + ). d Quantification of CM proliferation in wild-type sibling ( n = 6) and inhbaa OE ( n = 6) ventricles at 120 hpf. e Experimental setup of injections, EdU exposure, followed by fixation. f , g Tg(myl7:nlsDsRedExpress) hearts of myl7:H2B-EGFP and myl7:inhbaa-2A-H2B-EGFP(inhbaa OE) injected erbb −/− larvae at 120 hpf; α-DsRed (red), α-GFP (blue), EdU (green). White arrowheads point to proliferating CMs (EdU + /DsRed + /GFP + ). h Quantification of CM proliferation in myl7:H2B-EGFP ( n = 7) and inhbaa OE ( n = 7) injected ventricles at 120 hpf. i Experimental setup of EdU treatment, followed by fixation. j – m Tg(myl7:nlsDsRedExpress) hearts of wild-type sibling, inhbaa OE, nrg2a OE, and nrg2a OE/ inhbaa OE larvae at 120 hpf; α-DsRed (red), EdU (green). White arrowheads point to proliferating CMs (EdU + /DsRed + ). n Quantification of CM proliferation in wild-type sibling ( n = 4), inhbaa OE ( n = 4), nrg2a OE ( n = 4), and nrg2a OE/ inhbaa OE ( n = 5) ventricles at 120 hpf. o – r Tg(myl7:nlsDsRedExpress) hearts of wild-type sibling, inhbaa OE, mstnb OE, and mstnb OE/ inhbaa OE larvae at 120 hpf; α-DsRed (red), EdU (green). s Quantification of CM proliferation in wild-type sibling ( n = 5), inhbaa OE ( n = 4), mstnb OE ( n = 6), and mstnb OE/ inhbaa OE ( n = 6) ventricles at 120 hpf. All cell counts were performed on non-overlapping confocal planes (thickness, 1 µm) (data are mean ± s.e.m., ns: no significant changes observed, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001—Student’s t test, two-tailed). Scale bars, 20 µm. vent., ventricle; atr., atrium
    Erbb2 Signaling Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    ErbB2, Src, and STAT3 form a complex in ErbB2 overexpressing cells. Whole cell lysates from A. 435.ErbB2 or B. SKBR3 cells were immunoprecipitated (IP) with the indicated antibodies followed by immunoblot analysis for ErbB2, STAT3, and Src. Input lanes represent 10% of total protein immunoprecipitated. Asterisk indicates IgG heavy chain.

    Journal: Molecular cancer research : MCR

    Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells

    doi: 10.1158/1541-7786.MCR-08-0316

    Figure Lengend Snippet: ErbB2, Src, and STAT3 form a complex in ErbB2 overexpressing cells. Whole cell lysates from A. 435.ErbB2 or B. SKBR3 cells were immunoprecipitated (IP) with the indicated antibodies followed by immunoblot analysis for ErbB2, STAT3, and Src. Input lanes represent 10% of total protein immunoprecipitated. Asterisk indicates IgG heavy chain.

    Article Snippet: Role of erbB2 in breast cancer chemosensitivity.

    Techniques: Immunoprecipitation

    p21 Cip1 transcriptional activation by ErbB2 is dependant on STAT3 protein. A. 435.ErbB2 and 231.ErbB2 cells treated with 300 nM of Mismatch (MM) or STAT3 Antisense (AS) oligonucleotides. Cells were harvested 5 days post-transfection and analyzed by Western blot with the indicated antibodies. B. Standardized firefly luciferase activity of the wild type p21 promoter (pGL3-p21−2400) reporter plasmid in 435.ErbB2 and 231.ErbB2 cells treated with either STAT3 MM or AS oligonucleotides. Fold reduction was determined by standardizing to Mismatch control treated sample for each cell line. Error bars represent S.D.

    Journal: Molecular cancer research : MCR

    Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells

    doi: 10.1158/1541-7786.MCR-08-0316

    Figure Lengend Snippet: p21 Cip1 transcriptional activation by ErbB2 is dependant on STAT3 protein. A. 435.ErbB2 and 231.ErbB2 cells treated with 300 nM of Mismatch (MM) or STAT3 Antisense (AS) oligonucleotides. Cells were harvested 5 days post-transfection and analyzed by Western blot with the indicated antibodies. B. Standardized firefly luciferase activity of the wild type p21 promoter (pGL3-p21−2400) reporter plasmid in 435.ErbB2 and 231.ErbB2 cells treated with either STAT3 MM or AS oligonucleotides. Fold reduction was determined by standardizing to Mismatch control treated sample for each cell line. Error bars represent S.D.

    Article Snippet: Role of erbB2 in breast cancer chemosensitivity.

    Techniques: Activation Assay, Transfection, Western Blot, Luciferase, Activity Assay, Plasmid Preparation

    Inhibition of STAT3 sensitized ErbB2 overexpressing breast cancer cells to Taxol. A. 435.ErbB2 and SKBR3 cells were treated with 50 μM of STAT3 inhibitory peptide or control peptide for 24 hours. Nuclear extracts were then subjected to EMSA. Band shifts indicated a STAT3 protein:DNA complex. Oct-1 protein:DNA binding serves as an loading control. B. 435.ErbB2 and SKBR3 cells were treated with control (CP) or STAT3 inhibitor peptide (SP) (100 μM for 435.ErbB2 and 50 μM for SKBR3) for 12 hours. Cells were then treated with different concentrations of Taxol for 24 hours. Cell growth inhibition was determined by MTS assay. Error bars represent S.D. Inset: Representative Western blot for p21 Cip1 and β-actin (loading control) from cells treated with either control (CP) or STAT3 inhibitor peptide (SP) treatment (** p = 0.004; * p = 0.016; t-test).

    Journal: Molecular cancer research : MCR

    Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells

    doi: 10.1158/1541-7786.MCR-08-0316

    Figure Lengend Snippet: Inhibition of STAT3 sensitized ErbB2 overexpressing breast cancer cells to Taxol. A. 435.ErbB2 and SKBR3 cells were treated with 50 μM of STAT3 inhibitory peptide or control peptide for 24 hours. Nuclear extracts were then subjected to EMSA. Band shifts indicated a STAT3 protein:DNA complex. Oct-1 protein:DNA binding serves as an loading control. B. 435.ErbB2 and SKBR3 cells were treated with control (CP) or STAT3 inhibitor peptide (SP) (100 μM for 435.ErbB2 and 50 μM for SKBR3) for 12 hours. Cells were then treated with different concentrations of Taxol for 24 hours. Cell growth inhibition was determined by MTS assay. Error bars represent S.D. Inset: Representative Western blot for p21 Cip1 and β-actin (loading control) from cells treated with either control (CP) or STAT3 inhibitor peptide (SP) treatment (** p = 0.004; * p = 0.016; t-test).

    Article Snippet: Role of erbB2 in breast cancer chemosensitivity.

    Techniques: Inhibition, Binding Assay, MTS Assay, Western Blot

    Src is required for ErbB2 mediated STAT3 activation and p21 Cip1 upregulation. A. ErbB2 overexpressing cells were treated with 5 μM control (PP3) or Src inhibitor (PP2). Cell lysates were collected and analyzed by Western blotting. B. 435.ErbB2 and SKBR3 cells were transfected with the p21 Cip1 reporter plasmids wild type (pGL3-p21−2400) or SIE mutant (pGL3-p21−2400mSIE) and treated with 5 μM PP3 or PP2 for 24 hours. Cell lysates were collected and luciferase activities were measured and standardized by transfection efficiency using renilla values. Error bars represent S.D. C. Indicated cells were transfected with either vector control (Vec) or dominant negative Src mutant (pSRC-DN) for 24 hours. Lysates were analyzed by Western blotting. D. Cells were co-transfected with vector control or pSrc-DN plasmids and the pGL3-p21−2400 reporter plasmid. Luciferase activities were measured as in (C).

    Journal: Molecular cancer research : MCR

    Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells

    doi: 10.1158/1541-7786.MCR-08-0316

    Figure Lengend Snippet: Src is required for ErbB2 mediated STAT3 activation and p21 Cip1 upregulation. A. ErbB2 overexpressing cells were treated with 5 μM control (PP3) or Src inhibitor (PP2). Cell lysates were collected and analyzed by Western blotting. B. 435.ErbB2 and SKBR3 cells were transfected with the p21 Cip1 reporter plasmids wild type (pGL3-p21−2400) or SIE mutant (pGL3-p21−2400mSIE) and treated with 5 μM PP3 or PP2 for 24 hours. Cell lysates were collected and luciferase activities were measured and standardized by transfection efficiency using renilla values. Error bars represent S.D. C. Indicated cells were transfected with either vector control (Vec) or dominant negative Src mutant (pSRC-DN) for 24 hours. Lysates were analyzed by Western blotting. D. Cells were co-transfected with vector control or pSrc-DN plasmids and the pGL3-p21−2400 reporter plasmid. Luciferase activities were measured as in (C).

    Article Snippet: Role of erbB2 in breast cancer chemosensitivity.

    Techniques: Activation Assay, Western Blot, Transfection, Mutagenesis, Luciferase, Plasmid Preparation, Dominant Negative Mutation

    Inhibition of Src kinase sensitized ErbB2 overexpressing breast cancer cells to Taxol. A. 435.ErbB2 cells were treated as indicated for 18 hours. Cell lysates were collected and analyzed by western blot analysis for the indicated antibodies. Numbers represent relative intensity of each band compared to the non-treated (NT) control. Total STAT3 was used as the loading control for both P-STAT3-Y705 and p21. B. 435.ErbB2 cells were treated with DMSO (control) or AZD0530 for 18 hours. Chromatin Immunoprecipitation (ChIP assay) for SIE p21 Cip1 promoter sequence was performed using antibodies against histone and STAT3. IgG and Histone immunoprecipitation served as negative and positive controls, respectively. Input represents 10% of the total. C. 435.ErbB2 and SKBR3 cells were treated with DMSO only or AZD0530 (2 μM) for 12 hours. After 12 hours, cell media was replaced with medium containing either Taxol (50 nM) only, AZD0530 only or combination of Taxol and AZD0530. Cell growth inhibition was determined by MTS assay at 36 hours. Results were normalized to DMSO controls (** p = 0.002; * p = 0.013; t-test).

    Journal: Molecular cancer research : MCR

    Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells

    doi: 10.1158/1541-7786.MCR-08-0316

    Figure Lengend Snippet: Inhibition of Src kinase sensitized ErbB2 overexpressing breast cancer cells to Taxol. A. 435.ErbB2 cells were treated as indicated for 18 hours. Cell lysates were collected and analyzed by western blot analysis for the indicated antibodies. Numbers represent relative intensity of each band compared to the non-treated (NT) control. Total STAT3 was used as the loading control for both P-STAT3-Y705 and p21. B. 435.ErbB2 cells were treated with DMSO (control) or AZD0530 for 18 hours. Chromatin Immunoprecipitation (ChIP assay) for SIE p21 Cip1 promoter sequence was performed using antibodies against histone and STAT3. IgG and Histone immunoprecipitation served as negative and positive controls, respectively. Input represents 10% of the total. C. 435.ErbB2 and SKBR3 cells were treated with DMSO only or AZD0530 (2 μM) for 12 hours. After 12 hours, cell media was replaced with medium containing either Taxol (50 nM) only, AZD0530 only or combination of Taxol and AZD0530. Cell growth inhibition was determined by MTS assay at 36 hours. Results were normalized to DMSO controls (** p = 0.002; * p = 0.013; t-test).

    Article Snippet: Role of erbB2 in breast cancer chemosensitivity.

    Techniques: Inhibition, Western Blot, Chromatin Immunoprecipitation, Sequencing, Immunoprecipitation, MTS Assay

    Constitutive activation of STAT3 in ErbB2 overexpressing cells results in p21 Cip1 transcriptional upregulation. A. left , schematic diagram of the 5’ promoter region of the p21 gene. White boxes represent STAT SIE location on promoter; black box represents mutated SIE. right , Luciferase activity of extracts prepared from 435.Vec and 435.ErbB2 cells transfected with the indicated p21 Cip1 promoter reporter constructs. Bars indicate luciferase activity standardized to vector control for each construct. Error bars represent standard deviation (S.D.) B. Lysates from SKBR3, along with MDA-MB--435 and MDA-MB-231 breast cancer cells stably transfected with either control vector or wild-type ErbB2 were analyzed by Western blotting with the indicated antibodies. C. Nuclear extracts were collected from ErbB2 stable transfectants and vector control cells and SKBR3 cells for Electrophoretic Mobility Shift Assay (EMSA) analysis. Arrow indicates STAT protein:DNA complexes. Asterisk indicates addition of non-radiolabeled competitor probe. D. Chromatin Immunoprecipitation (ChIP assay) for SIE p21 Cip1 promoter sequence using antibodies against histone and STAT3 in ErbB2 overexpressing cells. Histone and IgG immunoprecipitation served as positive and negative controls, respectively. Input represents 10% of the total.

    Journal: Molecular cancer research : MCR

    Article Title: ErbB2-mediated Src and STAT3 Activation Leads to Transcriptional Upregulation of p21Cip1 and Chemoresistance in Breast Cancer Cells

    doi: 10.1158/1541-7786.MCR-08-0316

    Figure Lengend Snippet: Constitutive activation of STAT3 in ErbB2 overexpressing cells results in p21 Cip1 transcriptional upregulation. A. left , schematic diagram of the 5’ promoter region of the p21 gene. White boxes represent STAT SIE location on promoter; black box represents mutated SIE. right , Luciferase activity of extracts prepared from 435.Vec and 435.ErbB2 cells transfected with the indicated p21 Cip1 promoter reporter constructs. Bars indicate luciferase activity standardized to vector control for each construct. Error bars represent standard deviation (S.D.) B. Lysates from SKBR3, along with MDA-MB--435 and MDA-MB-231 breast cancer cells stably transfected with either control vector or wild-type ErbB2 were analyzed by Western blotting with the indicated antibodies. C. Nuclear extracts were collected from ErbB2 stable transfectants and vector control cells and SKBR3 cells for Electrophoretic Mobility Shift Assay (EMSA) analysis. Arrow indicates STAT protein:DNA complexes. Asterisk indicates addition of non-radiolabeled competitor probe. D. Chromatin Immunoprecipitation (ChIP assay) for SIE p21 Cip1 promoter sequence using antibodies against histone and STAT3 in ErbB2 overexpressing cells. Histone and IgG immunoprecipitation served as positive and negative controls, respectively. Input represents 10% of the total.

    Article Snippet: Role of erbB2 in breast cancer chemosensitivity.

    Techniques: Activation Assay, Luciferase, Activity Assay, Transfection, Construct, Plasmid Preparation, Standard Deviation, Multiple Displacement Amplification, Stable Transfection, Western Blot, Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, Sequencing, Immunoprecipitation

    Endogenous expression of plexinB1, Sema4D, ErbB2, and c-Met in primary prostate cells. A : Endogenous expression of indicated proteins in primary cultures of benign prostatic epithelium from three different individuals. B : Endogenous protein expression in immortalized benign prostatic epithelial cells from seven different individuals. AR, androgen receptor.

    Journal: The Prostate

    Article Title: Function of mutant and wild-type plexinB1 in prostate cancer cells

    doi: 10.1002/pros.22678

    Figure Lengend Snippet: Endogenous expression of plexinB1, Sema4D, ErbB2, and c-Met in primary prostate cells. A : Endogenous expression of indicated proteins in primary cultures of benign prostatic epithelium from three different individuals. B : Endogenous protein expression in immortalized benign prostatic epithelial cells from seven different individuals. AR, androgen receptor.

    Article Snippet: Furthermore, stimulation of LNCaP and LNCaP-LN3 with Sema4D resulted in phosphorylation of endogenous ErbB2 (Fig. B and D) and endogenous Akt (Fig. C and E), a signaling factor downstream of ErbB2.

    Techniques: Expressing

    PlexinB1-ErbB2 signaling in LNCaP and LNCaP-LN3 cells. A : PlexinB1 interacts with ErbB2 in LNCaP cells. Lysates of LNCaP were co-immunoprecipitated with plexinB1, ErbB2, or control antibody and pulled down proteins were detected by Western blotting. B : Sema4D treatment induces phosphorylation of endogenous ErbB2 in LNCaP cells. LNCaP cells were stimulated for 20 min with control or Sema4D conditioned medium or EGF (500 ng/ml). ErbB2 phosphorylation was detected by Western blotting. C : Sema4D treatment induces phosphorylation of Akt in LNCaP cells. LNCaP cells were stimulated for 20 min with control or Sema4D conditioned medium or EGF (500 ng/ml). Akt phosphorylation was detected by Western blotting ( i ) or on an antibody array ( ii and iii ). D : Sema4D treatment induces phosphorylation of endogenous ErbB2 in LNCaP-LN3 cells. LNCaP-LN3 cells were stimulated for 20 min with control or Sema4D conditioned medium or DHT (1 nM). ErbB2 phosphorylation was detected by Western blotting. E : Sema4D treatment induces phosphorylation of Akt in LNCaP-LN3 cells. LNCaP-LN3 cells were stimulated for 20 min with control or Sema4D conditioned medium or DHT (1 nM). Akt phosphorylation was detected by Western blotting. F : Knockdown of ErbB2 expression in LNCaP cells blocks Sema4D-induced migration. i : Transwell motility assay of LNCaP cells expressing non-silencing (NS) shRNA or two different shRNAs to ErbB2. Migration of Sema4D-stimulated cells relative to unstimulated cells (* P

    Journal: The Prostate

    Article Title: Function of mutant and wild-type plexinB1 in prostate cancer cells

    doi: 10.1002/pros.22678

    Figure Lengend Snippet: PlexinB1-ErbB2 signaling in LNCaP and LNCaP-LN3 cells. A : PlexinB1 interacts with ErbB2 in LNCaP cells. Lysates of LNCaP were co-immunoprecipitated with plexinB1, ErbB2, or control antibody and pulled down proteins were detected by Western blotting. B : Sema4D treatment induces phosphorylation of endogenous ErbB2 in LNCaP cells. LNCaP cells were stimulated for 20 min with control or Sema4D conditioned medium or EGF (500 ng/ml). ErbB2 phosphorylation was detected by Western blotting. C : Sema4D treatment induces phosphorylation of Akt in LNCaP cells. LNCaP cells were stimulated for 20 min with control or Sema4D conditioned medium or EGF (500 ng/ml). Akt phosphorylation was detected by Western blotting ( i ) or on an antibody array ( ii and iii ). D : Sema4D treatment induces phosphorylation of endogenous ErbB2 in LNCaP-LN3 cells. LNCaP-LN3 cells were stimulated for 20 min with control or Sema4D conditioned medium or DHT (1 nM). ErbB2 phosphorylation was detected by Western blotting. E : Sema4D treatment induces phosphorylation of Akt in LNCaP-LN3 cells. LNCaP-LN3 cells were stimulated for 20 min with control or Sema4D conditioned medium or DHT (1 nM). Akt phosphorylation was detected by Western blotting. F : Knockdown of ErbB2 expression in LNCaP cells blocks Sema4D-induced migration. i : Transwell motility assay of LNCaP cells expressing non-silencing (NS) shRNA or two different shRNAs to ErbB2. Migration of Sema4D-stimulated cells relative to unstimulated cells (* P

    Article Snippet: Furthermore, stimulation of LNCaP and LNCaP-LN3 with Sema4D resulted in phosphorylation of endogenous ErbB2 (Fig. B and D) and endogenous Akt (Fig. C and E), a signaling factor downstream of ErbB2.

    Techniques: Immunoprecipitation, Western Blot, Ab Array, Expressing, Migration, Motility Assay, shRNA

    Endogenous expression of plexinB1, Sema4D, ErbB2, and c-Met in prostate cancer cells. A : ( i ) Endogenous expression of plexinB1 and Sema4D mRNA in prostate cancer cell lines detected by RTPCR. ii : Endogenous expression of plexinB1 in prostate cancer cell lines detected by quantitative real time RTPCR, * P

    Journal: The Prostate

    Article Title: Function of mutant and wild-type plexinB1 in prostate cancer cells

    doi: 10.1002/pros.22678

    Figure Lengend Snippet: Endogenous expression of plexinB1, Sema4D, ErbB2, and c-Met in prostate cancer cells. A : ( i ) Endogenous expression of plexinB1 and Sema4D mRNA in prostate cancer cell lines detected by RTPCR. ii : Endogenous expression of plexinB1 in prostate cancer cell lines detected by quantitative real time RTPCR, * P

    Article Snippet: Furthermore, stimulation of LNCaP and LNCaP-LN3 with Sema4D resulted in phosphorylation of endogenous ErbB2 (Fig. B and D) and endogenous Akt (Fig. C and E), a signaling factor downstream of ErbB2.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Translocation of ErbB2 into mitochondria is kinase dependent. (A) The mitochondrial fractions and whole cell lysates were isolated and Western blotting was performed on MDA-MB-435ErbB2 (435eb), MDA-MB-435ErbB2V695E (435VE) and MDA-MB-435ErbB2K753M (435KM) cells. MtHSP70 and α-Tubulin were loading controls. (B) Mitochondrial fractions and whole cell lysates were isolated and Western Blotting was performed to detect mtErbB2 in mitochondria following the treatment of MCF7ErbB2 cells with ErbB2 inhibitor AG825 at 10 uM or control (DMSO) for 24 h; and with Heregulin β1 (HRG) at 10 ng/ml or control (PBS) for 24 h (upper). Whole cell lysates were subjected to Western Blotting to examine the phosphorylation status of ErbB2 and the total ErbB2. α-Tubulin was loading control (lower). (C) MCF7 cells were treated with and without HRG at 10 ng/ml for 24 h. The mitochondrial fraction and the whole cell lysate were isolated for Western blotting. Prohibitin and α-Tubulin were loading controls. (D) Left, mitochondrial proteins were isolated from SKBR3 cells and immunoprecipitated with anti-ErbB2 or anti-Cox II antibodies. The immunoprecipitates were analyzed by Western blotting with anti-ErbB2 and anti-Cox II antibodies. IgG was negative control. Right, mitochondrial proteins were isolated from 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells and immunoprecipited with Cox II antibody or negative control IgG. Samples were loaded on a SDS–PAGE followed by Western Blotting analysis with Cox II antibody to show the Cox II protein level, and with P-Y20 antibody to show the tyrosine phosphorylation level on Cox II. (E) Cytochrome c oxidase activities were measured after the cells were treated with or without a cytochrome c oxidase inhibitor Potassium cyanide (KCN) in 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells. The relative activities of Cox II were calculated relative to the Cox II activity of 231V cells. (F) 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells were treated with or without Taxol at 80 nM for 24 h. Cytosolic, mitochondrial fractions and whole cell lysates were analyzed by Western blotting with cytochrome c antibodies. α-Tubulin and mtHSP70 were cytosol and mitochondrial markers and loading controls. Columns , mean of three independent experiments; bars , SE.*, P

    Journal: Nature communications

    Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

    doi: 10.1038/ncomms2236

    Figure Lengend Snippet: Translocation of ErbB2 into mitochondria is kinase dependent. (A) The mitochondrial fractions and whole cell lysates were isolated and Western blotting was performed on MDA-MB-435ErbB2 (435eb), MDA-MB-435ErbB2V695E (435VE) and MDA-MB-435ErbB2K753M (435KM) cells. MtHSP70 and α-Tubulin were loading controls. (B) Mitochondrial fractions and whole cell lysates were isolated and Western Blotting was performed to detect mtErbB2 in mitochondria following the treatment of MCF7ErbB2 cells with ErbB2 inhibitor AG825 at 10 uM or control (DMSO) for 24 h; and with Heregulin β1 (HRG) at 10 ng/ml or control (PBS) for 24 h (upper). Whole cell lysates were subjected to Western Blotting to examine the phosphorylation status of ErbB2 and the total ErbB2. α-Tubulin was loading control (lower). (C) MCF7 cells were treated with and without HRG at 10 ng/ml for 24 h. The mitochondrial fraction and the whole cell lysate were isolated for Western blotting. Prohibitin and α-Tubulin were loading controls. (D) Left, mitochondrial proteins were isolated from SKBR3 cells and immunoprecipitated with anti-ErbB2 or anti-Cox II antibodies. The immunoprecipitates were analyzed by Western blotting with anti-ErbB2 and anti-Cox II antibodies. IgG was negative control. Right, mitochondrial proteins were isolated from 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells and immunoprecipited with Cox II antibody or negative control IgG. Samples were loaded on a SDS–PAGE followed by Western Blotting analysis with Cox II antibody to show the Cox II protein level, and with P-Y20 antibody to show the tyrosine phosphorylation level on Cox II. (E) Cytochrome c oxidase activities were measured after the cells were treated with or without a cytochrome c oxidase inhibitor Potassium cyanide (KCN) in 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells. The relative activities of Cox II were calculated relative to the Cox II activity of 231V cells. (F) 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells were treated with or without Taxol at 80 nM for 24 h. Cytosolic, mitochondrial fractions and whole cell lysates were analyzed by Western blotting with cytochrome c antibodies. α-Tubulin and mtHSP70 were cytosol and mitochondrial markers and loading controls. Columns , mean of three independent experiments; bars , SE.*, P

    Article Snippet: Vectors construction Vectors containing wild type ErbB2 and ErbB2 plus a mitochondrial targeting sequence (MTS) were constructed.

    Techniques: Translocation Assay, Isolation, Western Blot, Multiple Displacement Amplification, Immunoprecipitation, Negative Control, SDS Page, Activity Assay

    Localization of ErbB2 in mitochondria. (A) Cytosolic, nuclear, mitochondrial, and plasma membrane proteins were isolated and subjected to SDS-PAGE followed by probing with indicated antibodies. Two exogenous ErbB2 overexpressing breast cancer cell lines MCF7/ErbB2 and MDA-MB-231 (left), and two natural ErbB2-positive breast cancer cell lines SKBR3 and BT474 (right) were used for Western Blotting. VDAC1 and prohibitin were mitochondrial markers; Integrin β1 and IGF1Rα were plasma membrane markers; α-Tubulin and ERK were cytoplasmic markers; KDEL was an ER marker; EEA1 was an early endosomes marker; Golgi complex was a marker for the detection of Golgi; LAMP2 was an lysosome marker and Lamin B1 was a nucleus marker. (B) MCF7 cells, mouse heart and liver tissues, and ErbB2 positive and negative breast cancer patient samples were analyzed by Western blotting. (C) Co-localization of ErbB2 and mitochondria. Mitochondria were stained with Mitotracker-Green in ErbB2 transfected MCF7 cells. The cells were fixed and incubated with antibodies against ErbB2 (mouse), followed by incubation of monoclonal mouse Anti–Cy3 antibody (red). Images were analyzed with Nikon NIS-Elements AR software. Green: mitochondria; Red: ErbB2; Yellow: Co-localization of ErbB2 and mitochondria. The lower panel contains images with a higher magnification. Scale bars: 20 μm. (D) Localization of ErbB2 inside mitochondria. Intact mitochondria of SKBR3 cells were isolated and treated with Protease K at 0.5 mg/ml for 15 min at room temperature. 1% Triton was added into the reaction and 2 mM PMSF was added to stop the reaction. Each reaction was started with an equal amount/volume of mitochondria lysate. Mitochondrial lysate then was loaded onto 8% SDS PAGE followed by blotting with anti-ErbB2, anti-BCL-2 and anti-Prohibitin antibodies.

    Journal: Nature communications

    Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

    doi: 10.1038/ncomms2236

    Figure Lengend Snippet: Localization of ErbB2 in mitochondria. (A) Cytosolic, nuclear, mitochondrial, and plasma membrane proteins were isolated and subjected to SDS-PAGE followed by probing with indicated antibodies. Two exogenous ErbB2 overexpressing breast cancer cell lines MCF7/ErbB2 and MDA-MB-231 (left), and two natural ErbB2-positive breast cancer cell lines SKBR3 and BT474 (right) were used for Western Blotting. VDAC1 and prohibitin were mitochondrial markers; Integrin β1 and IGF1Rα were plasma membrane markers; α-Tubulin and ERK were cytoplasmic markers; KDEL was an ER marker; EEA1 was an early endosomes marker; Golgi complex was a marker for the detection of Golgi; LAMP2 was an lysosome marker and Lamin B1 was a nucleus marker. (B) MCF7 cells, mouse heart and liver tissues, and ErbB2 positive and negative breast cancer patient samples were analyzed by Western blotting. (C) Co-localization of ErbB2 and mitochondria. Mitochondria were stained with Mitotracker-Green in ErbB2 transfected MCF7 cells. The cells were fixed and incubated with antibodies against ErbB2 (mouse), followed by incubation of monoclonal mouse Anti–Cy3 antibody (red). Images were analyzed with Nikon NIS-Elements AR software. Green: mitochondria; Red: ErbB2; Yellow: Co-localization of ErbB2 and mitochondria. The lower panel contains images with a higher magnification. Scale bars: 20 μm. (D) Localization of ErbB2 inside mitochondria. Intact mitochondria of SKBR3 cells were isolated and treated with Protease K at 0.5 mg/ml for 15 min at room temperature. 1% Triton was added into the reaction and 2 mM PMSF was added to stop the reaction. Each reaction was started with an equal amount/volume of mitochondria lysate. Mitochondrial lysate then was loaded onto 8% SDS PAGE followed by blotting with anti-ErbB2, anti-BCL-2 and anti-Prohibitin antibodies.

    Article Snippet: Vectors construction Vectors containing wild type ErbB2 and ErbB2 plus a mitochondrial targeting sequence (MTS) were constructed.

    Techniques: Isolation, SDS Page, Multiple Displacement Amplification, Western Blot, Marker, Staining, Transfection, Incubation, Software

    Analysis of the mechanisms of mtErbB2 mitochondrial localization. (A) MCF7 breast cancer cells were transfected with plasmids encoding either GFP alone, or GFP fused ErbB2 fragments GFP-646–689, GFP-623–645 or GFP-623–689. Cells were cultured and the florescent imaging was done as described under the “Methods.” Scale bars: 20 μm. (B) MDA-MB-231 cells were transfected with wild-type ErbB2 and ErbB2ΔMTS vectors and mitochondrial proteins were extracted. ErbB2 expression was measured by Western blotting analysis; α-Tubulin and mtHSP70 were loading controls. (C) Mitochondrial proteins were isolated from MDA-MB-231ErbB2 cells and immunoprecipitated with ErbB2 antibody. The immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top left). Mitochondrial proteins from the same cells were precipitated with mtHSP70 antibody and the immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top right). IgG was used as a negative control. Isolated mitochondrial proteins (input) were loaded as a positive control. Similar results were obtained using another breast cancer cell line, SKBR3 (bottom). (D) siRNA specific to mtHSP70 was transfected into MDA-MB-231ErbB2 cells. The cytoplasmic fraction (Cyto), mitochondrial fraction (Mito) and whole cell lysate (WCL) were separated for western blotting analysis (left). Cytochrome c oxidase subunit II and α-Tubulin were makers and loading controls for the mitochondrial fraction and the cytoplasmic fraction, respectively. The relative protein amounts of ErbB2 and mtHSP70 were calculated by determining the intensity of the protein bands followed by the normalization with loading controls (right). Experiments were repeated three times. Columns, mean of three independent experiments; bars, SE.

    Journal: Nature communications

    Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

    doi: 10.1038/ncomms2236

    Figure Lengend Snippet: Analysis of the mechanisms of mtErbB2 mitochondrial localization. (A) MCF7 breast cancer cells were transfected with plasmids encoding either GFP alone, or GFP fused ErbB2 fragments GFP-646–689, GFP-623–645 or GFP-623–689. Cells were cultured and the florescent imaging was done as described under the “Methods.” Scale bars: 20 μm. (B) MDA-MB-231 cells were transfected with wild-type ErbB2 and ErbB2ΔMTS vectors and mitochondrial proteins were extracted. ErbB2 expression was measured by Western blotting analysis; α-Tubulin and mtHSP70 were loading controls. (C) Mitochondrial proteins were isolated from MDA-MB-231ErbB2 cells and immunoprecipitated with ErbB2 antibody. The immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top left). Mitochondrial proteins from the same cells were precipitated with mtHSP70 antibody and the immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top right). IgG was used as a negative control. Isolated mitochondrial proteins (input) were loaded as a positive control. Similar results were obtained using another breast cancer cell line, SKBR3 (bottom). (D) siRNA specific to mtHSP70 was transfected into MDA-MB-231ErbB2 cells. The cytoplasmic fraction (Cyto), mitochondrial fraction (Mito) and whole cell lysate (WCL) were separated for western blotting analysis (left). Cytochrome c oxidase subunit II and α-Tubulin were makers and loading controls for the mitochondrial fraction and the cytoplasmic fraction, respectively. The relative protein amounts of ErbB2 and mtHSP70 were calculated by determining the intensity of the protein bands followed by the normalization with loading controls (right). Experiments were repeated three times. Columns, mean of three independent experiments; bars, SE.

    Article Snippet: Vectors construction Vectors containing wild type ErbB2 and ErbB2 plus a mitochondrial targeting sequence (MTS) were constructed.

    Techniques: Transfection, Cell Culture, Imaging, Multiple Displacement Amplification, Expressing, Western Blot, Isolation, Immunoprecipitation, Negative Control, Positive Control

    Translocation of ErbB2 into mitochondria contributes to trastuzumab resistance. (A) mtErbB2 is elevated in trastuzumab-resistant cancer cells. Mitochondrial proteins and whole cell lysates of BT474 and BT474 trastuzumab-resistant cells (BT474 HCP R) were isolated and analyzed by Western blotting. ATP Synthase and α-Tubulin were used as loading controls. (B) BT474 cells were treated with trastuzumab (HCP) at 10 ug/ml for 24 h and 48 h followed by the separation of cytosolic and mitochondrial fractions. Proteins from the cytoplasm, mitochondria and whole cell lysates were loaded onto gels and analyzed by Western blotting. mtHSP70 and α-Tubulin were used as loading controls (left). The relative protein amount of ErbB2 in the cellular fractions was calculated by detecting the intensity of the protein bands followed by normalization with loading controls (right). The experiments were repeated for three times. (C) mtErbB2-overexpressing cells are more resistant to trastuzumab. 231ErbB2WT, 231ErbB2Mito, 231ErbB2ΔMTS, 231ErbB2MitoKM and 231ErbB2WT cells transfected by siRNA to mtHSP70 were treated with trastuzumab at 100 ug/ml for 48 h and 72 h. The cell growth inhibition ratios were detected by a CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit. Columns, mean of three independent experiments; bars, SE. *, P

    Journal: Nature communications

    Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

    doi: 10.1038/ncomms2236

    Figure Lengend Snippet: Translocation of ErbB2 into mitochondria contributes to trastuzumab resistance. (A) mtErbB2 is elevated in trastuzumab-resistant cancer cells. Mitochondrial proteins and whole cell lysates of BT474 and BT474 trastuzumab-resistant cells (BT474 HCP R) were isolated and analyzed by Western blotting. ATP Synthase and α-Tubulin were used as loading controls. (B) BT474 cells were treated with trastuzumab (HCP) at 10 ug/ml for 24 h and 48 h followed by the separation of cytosolic and mitochondrial fractions. Proteins from the cytoplasm, mitochondria and whole cell lysates were loaded onto gels and analyzed by Western blotting. mtHSP70 and α-Tubulin were used as loading controls (left). The relative protein amount of ErbB2 in the cellular fractions was calculated by detecting the intensity of the protein bands followed by normalization with loading controls (right). The experiments were repeated for three times. (C) mtErbB2-overexpressing cells are more resistant to trastuzumab. 231ErbB2WT, 231ErbB2Mito, 231ErbB2ΔMTS, 231ErbB2MitoKM and 231ErbB2WT cells transfected by siRNA to mtHSP70 were treated with trastuzumab at 100 ug/ml for 48 h and 72 h. The cell growth inhibition ratios were detected by a CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit. Columns, mean of three independent experiments; bars, SE. *, P

    Article Snippet: Vectors construction Vectors containing wild type ErbB2 and ErbB2 plus a mitochondrial targeting sequence (MTS) were constructed.

    Techniques: Translocation Assay, Isolation, Western Blot, Transfection, Inhibition, Proliferation Assay

    inhbaa GOF promotes CM proliferation independently of ErbB2 signaling and competes with m stnb GOF. a Experimental setup of EdU treatment, followed by fixation. b , c Tg(myl7:nlsDsRedExpress) hearts of wild-type sibling and inhbaa OE larvae at 120 hpf; α-DsRed (red), EdU (green). White arrowheads point to proliferating CMs (EdU + /DsRed + ). d Quantification of CM proliferation in wild-type sibling ( n = 6) and inhbaa OE ( n = 6) ventricles at 120 hpf. e Experimental setup of injections, EdU exposure, followed by fixation. f , g Tg(myl7:nlsDsRedExpress) hearts of myl7:H2B-EGFP and myl7:inhbaa-2A-H2B-EGFP(inhbaa OE) injected erbb −/− larvae at 120 hpf; α-DsRed (red), α-GFP (blue), EdU (green). White arrowheads point to proliferating CMs (EdU + /DsRed + /GFP + ). h Quantification of CM proliferation in myl7:H2B-EGFP ( n = 7) and inhbaa OE ( n = 7) injected ventricles at 120 hpf. i Experimental setup of EdU treatment, followed by fixation. j – m Tg(myl7:nlsDsRedExpress) hearts of wild-type sibling, inhbaa OE, nrg2a OE, and nrg2a OE/ inhbaa OE larvae at 120 hpf; α-DsRed (red), EdU (green). White arrowheads point to proliferating CMs (EdU + /DsRed + ). n Quantification of CM proliferation in wild-type sibling ( n = 4), inhbaa OE ( n = 4), nrg2a OE ( n = 4), and nrg2a OE/ inhbaa OE ( n = 5) ventricles at 120 hpf. o – r Tg(myl7:nlsDsRedExpress) hearts of wild-type sibling, inhbaa OE, mstnb OE, and mstnb OE/ inhbaa OE larvae at 120 hpf; α-DsRed (red), EdU (green). s Quantification of CM proliferation in wild-type sibling ( n = 5), inhbaa OE ( n = 4), mstnb OE ( n = 6), and mstnb OE/ inhbaa OE ( n = 6) ventricles at 120 hpf. All cell counts were performed on non-overlapping confocal planes (thickness, 1 µm) (data are mean ± s.e.m., ns: no significant changes observed, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001—Student’s t test, two-tailed). Scale bars, 20 µm. vent., ventricle; atr., atrium

    Journal: Nature Communications

    Article Title: Opposite effects of Activin type 2 receptor ligands on cardiomyocyte proliferation during development and repair

    doi: 10.1038/s41467-017-01950-1

    Figure Lengend Snippet: inhbaa GOF promotes CM proliferation independently of ErbB2 signaling and competes with m stnb GOF. a Experimental setup of EdU treatment, followed by fixation. b , c Tg(myl7:nlsDsRedExpress) hearts of wild-type sibling and inhbaa OE larvae at 120 hpf; α-DsRed (red), EdU (green). White arrowheads point to proliferating CMs (EdU + /DsRed + ). d Quantification of CM proliferation in wild-type sibling ( n = 6) and inhbaa OE ( n = 6) ventricles at 120 hpf. e Experimental setup of injections, EdU exposure, followed by fixation. f , g Tg(myl7:nlsDsRedExpress) hearts of myl7:H2B-EGFP and myl7:inhbaa-2A-H2B-EGFP(inhbaa OE) injected erbb −/− larvae at 120 hpf; α-DsRed (red), α-GFP (blue), EdU (green). White arrowheads point to proliferating CMs (EdU + /DsRed + /GFP + ). h Quantification of CM proliferation in myl7:H2B-EGFP ( n = 7) and inhbaa OE ( n = 7) injected ventricles at 120 hpf. i Experimental setup of EdU treatment, followed by fixation. j – m Tg(myl7:nlsDsRedExpress) hearts of wild-type sibling, inhbaa OE, nrg2a OE, and nrg2a OE/ inhbaa OE larvae at 120 hpf; α-DsRed (red), EdU (green). White arrowheads point to proliferating CMs (EdU + /DsRed + ). n Quantification of CM proliferation in wild-type sibling ( n = 4), inhbaa OE ( n = 4), nrg2a OE ( n = 4), and nrg2a OE/ inhbaa OE ( n = 5) ventricles at 120 hpf. o – r Tg(myl7:nlsDsRedExpress) hearts of wild-type sibling, inhbaa OE, mstnb OE, and mstnb OE/ inhbaa OE larvae at 120 hpf; α-DsRed (red), EdU (green). s Quantification of CM proliferation in wild-type sibling ( n = 5), inhbaa OE ( n = 4), mstnb OE ( n = 6), and mstnb OE/ inhbaa OE ( n = 6) ventricles at 120 hpf. All cell counts were performed on non-overlapping confocal planes (thickness, 1 µm) (data are mean ± s.e.m., ns: no significant changes observed, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001—Student’s t test, two-tailed). Scale bars, 20 µm. vent., ventricle; atr., atrium

    Article Snippet: ErBb2 and TGF-β signaling inhibitor treatments The ErBb2 signaling inhibitor (PD168393, Calbiochem) , Smad3 phosphorylation inhibitor (SIS3, Calbiochem) and Activin type 1 receptor inhibitor (SB431542, Calbiochem) were used to treat the embryos or larvae.

    Techniques: Injection, Two Tailed Test