mthsp70 proteins was performed using respective primary antibodies  (Thermo Fisher)

Journal: Antioxidants & Redox Signaling

Article Title: Selective Disruption of Respiratory Supercomplexes as a New Strategy to Suppress Her2 high Breast Cancer

doi: 10.1089/ars.2016.6677

Figure Lengend Snippet: Her2 is localized at the inner mitochondrial membrane. (A) MCF7 Her2high cells stained using anti-ATPβ IgG, followed by Alexa Fluor 488-stained secondary IgG, and anti-Her2 IgG, followed by Alexa Fluor 555 secondary IgG, were inspected by STED confocal microscopy. White arrows show colocalization of anti-ATPβ and anti-Her2 signals. Size bar = 5 μm. (B) Breast cancer cell lines as shown were fractionated into the cytosolic + plasma membrane fraction and the mitochondrial fraction and assessed for the level of Her2 by WB following SDS-PAGE. Mitochondrial marker, COXIV, and cytosolic markers, SOD1 and actin, were used as loading controls. (C) MCF7 and MCF7 Her2high cells were assessed for localization of Her2 using IG-TEM. The light blue arrowheads show position of gold particles associated with Her2 in mitochondria, the green ones outside mitochondria, often pointing to stress fibers. Size bar = 0.5 μm. The two images on the right-hand side are enlarged boxed regions in the middle images. (D) MCF7 Her2high cells were subjected to double-staining with anti-Her2 IgG, followed by Alexa Fluor 647-stained secondary IgG, and anti-mtHsp70 IgG, followed by Cy3b-stained secondary IgG, and inspected using the super-resolution PALM microscopy. The left and bottom images are enlarged boxed images in the top right-hand micrograph. Size bar = 10 μm. (E) MCF7 Her2high cell lysate, their cytosolic + plasma membrane fraction (C+PMF), mitochondria, mitoplasts, and intermembrane space + outer membrane (IMS+OMM) fraction were assessed for Her2 using WB after SDS-PAGE with actin, NDUFA9, VDAC, Cyt c, and SDHA as loading controls and preparation markers. (F) Mitochondrial fraction of MCF7 Her2high cells was exposed to trypsin in the absence or presence of Triton X-100 for the periods indicated, at which time the preparations were assessed for Her2 by WB following SDS-PAGE. VDAC, ATPβ, NDUFS3, COXIV, and Cyt c were used as markers of different mitochondrial compartments. (G) Cytosolic + plasma membrane fraction and mitochondria of MCF7 mock or MCF7 Her2high tumors, excised from either control or MitoTam-exposed mice, were evaluated for Her2 by WB following SDS-PAGE with COXIV and actin as fraction markers and loading controls. All images represent at least three independent experiments. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: Immunocytochemistry of Her2 and mtHSP70 proteins was performed using respective primary antibodies and secondary antibodies conjugated with Alexa Fluor 647 and Cy3b (Life Technologies).

Techniques: Staining, Confocal Microscopy, SDS Page, Marker, Double Staining, Microscopy

Journal: Antioxidants & Redox Signaling

Article Title: Selective Disruption of Respiratory Supercomplexes as a New Strategy to Suppress Her2 high Breast Cancer

doi: 10.1089/ars.2016.6677

Figure Lengend Snippet: Mitochondrial fraction of Her2 determines sensitivity to MitoTam. (A) Mitochondrial fractions of MCF7 Her2high and BT474 cells were immunoprecipitated with anti-Her2 IgG and the immunoprecipitate, input, and flow-through inspected for Her2 and mtHsp70 by WB after SDS-PAGE. (B) MCF7 Her2high cells transfected with mtHSP70 siRNA or NS siRNA were assessed for Her2 using qPCR. (C) MCF7, MCF7 Her2low, and MCF7 Her2high cells were transfected with siRNA against mtHSP70 or with NS siRNA, left to recover for 24 h, and then exposed to 2 μM MitoTam for 24 h and assessed for cell death. (D) MCF7 Her2high cells were transfected with mtHSP70 siRNA or NS siRNA and the whole cell lysates were assessed for Her2, NDUFA9, and mtHSP70 by WB; Actin was used as a loading control. (E) MCF7 Her2high cells were transfected with mtHSP70 siRNA or NS siRNA, and cytoplasmic and mitochondrial fractions were assessed for Her2 and NDUFA9 by WB. VDAC and tubulin-α were used as a loading controls. (F) MCF7 Her2high cells were transfected with mtHSP70 siRNA or NS siRNA, and the solubilized mitochondria were assessed for NDUFA9, SDHA (probed after NDUFA9 using the same membrane), and UQCRC2 by WB after NBGE. VDAC was used as a loading control. (G) MCF7 cells transiently transfected with empty vector, wild-type Her2, Her2-MTS, or Her2-ΔMTS were exposed to 2 μM MitoTam for 24 h and assessed for cell death. Insert shows the protein level of Her2 in mitochondrial fraction in MTS- and ΔMTS-transfected cells. SDHB was used as a loading control. (H) Whole cell lysates of transiently transfected cells show an even level of Her2. Actin and VDAC were used as loading controls. The symbol, *, indicates statistically significant difference between cells transfected with NS and mtHSP70 siRNA (B), between MCF7, MCF7 Her2high, and MCF7 Her2low cells transfected with NS or mtHsp70 and MCF7 Her2high cells transfected with NS and mtHsp70 cells (C), and MCF7 cells transfected with wt, MTS, or ΔMTS plasmids (G). Images (A, D–H) are representatives of at least three independent experiments. MTS, mitochondrial targeting sequence.

Article Snippet: Immunocytochemistry of Her2 and mtHSP70 proteins was performed using respective primary antibodies and secondary antibodies conjugated with Alexa Fluor 647 and Cy3b (Life Technologies).

Techniques: Immunoprecipitation, SDS Page, Transfection, Plasmid Preparation, Sequencing

Journal: Antioxidants & Redox Signaling

Article Title: Selective Disruption of Respiratory Supercomplexes as a New Strategy to Suppress Her2 high Breast Cancer

doi: 10.1089/ars.2016.6677

Figure Lengend Snippet: IC 50 Values for Killing of Breast Cancer Cell Lines and Nonmalignant Cells with Tamoxifen and MitoTam

Article Snippet: Immunocytochemistry of Her2 and mtHSP70 proteins was performed using respective primary antibodies and secondary antibodies conjugated with Alexa Fluor 647 and Cy3b (Life Technologies).

Techniques:

Journal: Antioxidants & Redox Signaling

Article Title: Selective Disruption of Respiratory Supercomplexes as a New Strategy to Suppress Her2 high Breast Cancer

doi: 10.1089/ars.2016.6677

Figure Lengend Snippet: MitoTam is more efficient in killing Her2high cells than their Her2low counterparts. (A) MCF7 parental, Her2low (shRNA transfected), mock (empty plasmid transfected), and Her2high cells (Her2 plasmid transfected) and MDA-MB-231 parental, mock, and Her2high cells were assessed for the Her2 protein in whole cell lysate using WB with actin as a loading control. MCF7, MCF7 Her2low, and MCF7 Her2high cells were exposed to (B) tamoxifen or (C) MitoTam at the concentrations shown for 16 h and cell viability assessed using the crystal violet method. (D) MCF7, MCF7 Her2low, and MCF7 Her2high cells and (E) MDA-MB-231, MDA-MB-231 mock, and MDA-MB-231 Her2high cells were exposed to MitoTam at the concentrations shown for 24 h, and cell death was evaluated using Annexin V/PI staining. (F) MCF7, MCF7 Her2low, and MCF7 Her2high cells were exposed to TAM-DPPO at the concentrations shown for 24 h and cell death was evaluated using Annexin V/PI staining. (G) MCF7 mock and MCF7 Her2high cells were exposed to 2.5 μM MitoTam for the times shown and the levels of procaspase-9, caspase-9, Parp 1/2 (both the intact and cleaved forms, indicated by arrows), Bax, Bcl2, and Her2, with actin as loading control, were estimated by WB. (H) MCF7, MCF7 mock, and MCF7 Her2high cells were seeded in Petri dishes in soft agar, cultured for 14 days after 16-h treatment with 20 μM tamoxifen or 2.5 μM MitoTam, and stained with crystal violet to visualize individual colonies. (I) MCF7 and (J) MCF7 Her2high cells were exposed to 2.5 μM MitoTam or solvent control in the presence of lapatinib (0.5 μM) or mubritinib (0.5 μM) for 24 h and cell death was evaluated. Images in (A), (G), and (H) are representative of three independent experiments. Data in all other panels are mean values (n ≥ 3) ± SEM. The symbol, *, indicates statistically significant differences (p < 0.05).

Article Snippet: Immunocytochemistry of Her2 and mtHSP70 proteins was performed using respective primary antibodies and secondary antibodies conjugated with Alexa Fluor 647 and Cy3b (Life Technologies).

Techniques: shRNA, Transfection, Plasmid Preparation, Staining, Cell Culture

Journal: Antioxidants & Redox Signaling

Article Title: Selective Disruption of Respiratory Supercomplexes as a New Strategy to Suppress Her2 high Breast Cancer

doi: 10.1089/ars.2016.6677

Figure Lengend Snippet: MitoTam efficiently suppresses Her2high breast carcinomas. (A) FVB/N c-neu mice s.c. injected with syngeneic NeuTL cells (2 × 106 cells per animal) were treated twice a week with tamoxifen (2.69 μmol/mouse/dose) or MitoTam (0.54 μmol/mouse/dose) dissolved in 4% EtOH in corn oil, 100 μl per dose, and tumor volume evaluated by USI. (B) Balb-c nu/nu mice were implanted with a slow-release estradiol pellet and injected s.c. with 2 × 106 MCF7 mock or MCF7 Her2high cells per animal. As soon as USI-detectable tumors appeared (∼50 mm3), the mice were treated with i.p. injection with 100 μl of tamoxifen (2 μmol/mouse/dose) or MitoTam (0.25 μmol/mouse/dose) dissolved in 4% EtOH in corn oil on days 3 and 7 of every week, and tumor volume was visualized and evaluated using USI. (C) Control and MitoTam-treated MCF7 mock and MCF7 Her2high cell-derived tumors, excised at the end of the experiment, were fixed, paraffin embedded, and stained using the TUNEL technique. The arrows indicate TUNEL-positive cells. Size bar = 50 μm. (D) Control and treated tumors as shown in (B) were lysed and evaluated for the level of procaspase-9, caspase-9, Bax, Bcl-2, and Her2 with actin as loading control using WB. (E) FVB/N c-neu mice with spontaneous tumors were treated twice a week with MitoTam (0.54 μmol/mouse/dose) or solvent control, and tumor volume was evaluated. (F) Balb/c mice were s.c. injected with syngeneic 4T1 cells (1 × 106 cells per animal) and treated with MitoTam (0.25 μmol/mouse/dose) or solvent control twice a week, and tumor volume was evaluated. (G) Blood, lung, and liver harvested from animals in (F) were homogenized and subjected to selection in the presence of 6-thioguanine for 10 days and colonies were counted. Data in (A) and (B) are mean values (n = 6) ± SEM and, in (E) and (F), are mean values (n ≥ 5) ± SEM. The symbols, *, **, and #, indicate statistically significant differences (p < 0.05). WB, Western blotting. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: Immunocytochemistry of Her2 and mtHSP70 proteins was performed using respective primary antibodies and secondary antibodies conjugated with Alexa Fluor 647 and Cy3b (Life Technologies).

Techniques: Injection, Derivative Assay, Staining, TUNEL Assay, Selection, Western Blot

Journal: Antioxidants & Redox Signaling

Article Title: Selective Disruption of Respiratory Supercomplexes as a New Strategy to Suppress Her2 high Breast Cancer

doi: 10.1089/ars.2016.6677

Figure Lengend Snippet: Her2 is localized at the inner mitochondrial membrane. (A) MCF7 Her2high cells stained using anti-ATPβ IgG, followed by Alexa Fluor 488-stained secondary IgG, and anti-Her2 IgG, followed by Alexa Fluor 555 secondary IgG, were inspected by STED confocal microscopy. White arrows show colocalization of anti-ATPβ and anti-Her2 signals. Size bar = 5 μm. (B) Breast cancer cell lines as shown were fractionated into the cytosolic + plasma membrane fraction and the mitochondrial fraction and assessed for the level of Her2 by WB following SDS-PAGE. Mitochondrial marker, COXIV, and cytosolic markers, SOD1 and actin, were used as loading controls. (C) MCF7 and MCF7 Her2high cells were assessed for localization of Her2 using IG-TEM. The light blue arrowheads show position of gold particles associated with Her2 in mitochondria, the green ones outside mitochondria, often pointing to stress fibers. Size bar = 0.5 μm. The two images on the right-hand side are enlarged boxed regions in the middle images. (D) MCF7 Her2high cells were subjected to double-staining with anti-Her2 IgG, followed by Alexa Fluor 647-stained secondary IgG, and anti-mtHsp70 IgG, followed by Cy3b-stained secondary IgG, and inspected using the super-resolution PALM microscopy. The left and bottom images are enlarged boxed images in the top right-hand micrograph. Size bar = 10 μm. (E) MCF7 Her2high cell lysate, their cytosolic + plasma membrane fraction (C+PMF), mitochondria, mitoplasts, and intermembrane space + outer membrane (IMS+OMM) fraction were assessed for Her2 using WB after SDS-PAGE with actin, NDUFA9, VDAC, Cyt c, and SDHA as loading controls and preparation markers. (F) Mitochondrial fraction of MCF7 Her2high cells was exposed to trypsin in the absence or presence of Triton X-100 for the periods indicated, at which time the preparations were assessed for Her2 by WB following SDS-PAGE. VDAC, ATPβ, NDUFS3, COXIV, and Cyt c were used as markers of different mitochondrial compartments. (G) Cytosolic + plasma membrane fraction and mitochondria of MCF7 mock or MCF7 Her2high tumors, excised from either control or MitoTam-exposed mice, were evaluated for Her2 by WB following SDS-PAGE with COXIV and actin as fraction markers and loading controls. All images represent at least three independent experiments. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: Immunocytochemistry of Her2 and mtHSP70 proteins was performed using respective primary antibodies and secondary antibodies conjugated with Alexa Fluor 647 and Cy3b (Life Technologies).

Techniques: Staining, Confocal Microscopy, SDS Page, Marker, Double Staining, Microscopy

Journal: Antioxidants & Redox Signaling

Article Title: Selective Disruption of Respiratory Supercomplexes as a New Strategy to Suppress Her2 high Breast Cancer

doi: 10.1089/ars.2016.6677

Figure Lengend Snippet: Mitochondrial fraction of Her2 determines sensitivity to MitoTam. (A) Mitochondrial fractions of MCF7 Her2high and BT474 cells were immunoprecipitated with anti-Her2 IgG and the immunoprecipitate, input, and flow-through inspected for Her2 and mtHsp70 by WB after SDS-PAGE. (B) MCF7 Her2high cells transfected with mtHSP70 siRNA or NS siRNA were assessed for Her2 using qPCR. (C) MCF7, MCF7 Her2low, and MCF7 Her2high cells were transfected with siRNA against mtHSP70 or with NS siRNA, left to recover for 24 h, and then exposed to 2 μM MitoTam for 24 h and assessed for cell death. (D) MCF7 Her2high cells were transfected with mtHSP70 siRNA or NS siRNA and the whole cell lysates were assessed for Her2, NDUFA9, and mtHSP70 by WB; Actin was used as a loading control. (E) MCF7 Her2high cells were transfected with mtHSP70 siRNA or NS siRNA, and cytoplasmic and mitochondrial fractions were assessed for Her2 and NDUFA9 by WB. VDAC and tubulin-α were used as a loading controls. (F) MCF7 Her2high cells were transfected with mtHSP70 siRNA or NS siRNA, and the solubilized mitochondria were assessed for NDUFA9, SDHA (probed after NDUFA9 using the same membrane), and UQCRC2 by WB after NBGE. VDAC was used as a loading control. (G) MCF7 cells transiently transfected with empty vector, wild-type Her2, Her2-MTS, or Her2-ΔMTS were exposed to 2 μM MitoTam for 24 h and assessed for cell death. Insert shows the protein level of Her2 in mitochondrial fraction in MTS- and ΔMTS-transfected cells. SDHB was used as a loading control. (H) Whole cell lysates of transiently transfected cells show an even level of Her2. Actin and VDAC were used as loading controls. The symbol, *, indicates statistically significant difference between cells transfected with NS and mtHSP70 siRNA (B), between MCF7, MCF7 Her2high, and MCF7 Her2low cells transfected with NS or mtHsp70 and MCF7 Her2high cells transfected with NS and mtHsp70 cells (C), and MCF7 cells transfected with wt, MTS, or ΔMTS plasmids (G). Images (A, D–H) are representatives of at least three independent experiments. MTS, mitochondrial targeting sequence.

Article Snippet: Immunocytochemistry of Her2 and mtHSP70 proteins was performed using respective primary antibodies and secondary antibodies conjugated with Alexa Fluor 647 and Cy3b (Life Technologies).

Techniques: Immunoprecipitation, SDS Page, Transfection, Plasmid Preparation, Sequencing