sc205346  (Santa Cruz Biotechnology)

Journal: Molecular and Cellular Biology

Article Title: Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α

doi: 10.1128/MCB.00180-16

Figure Lengend Snippet: HIF-1α mediates CXCR4 induction by HRG in breast cancer cells. (A) Induction of HIF-1α by HRG. BT-474, MDA-MB-361, and MCF-7 breast cancer cells were incubated with HRG (10 ng/ml) for different times. HIF-1α, phospho-Akt, and phospho-Erk were determined by Western blotting in cell lysates. (Top) Short-term incubation with HRG. (Bottom) Long-term incubation with HRG. As a positive control for HIF-1α induction, CoCl2 (100 μM) was used. (B) HRG (10 ng/ml) does not induce HIF-1α in MCF-10A cells. (C) Breast cancer cells were incubated with HRG (10 ng/ml; 16 h) or vehicle (control). After washing, cells were lysed at different times (0 to 8 h), and HIF-1α expression was determined by Western blotting. (D) Effect of the HIF-1α inhibitor SC205346 (0.3 to 100 μM, added 1 h before and during HRG treatment) on HIF-1α induction by HRG (10 ng/ml; 6 h) in MCF-7 cells. (Left) Representative experiment. (Right) Densitometric values of HIF-1α expression levels (means and SEM of the results of 3 independent experiments) expressed as percentages relative to cells with HRG treatment. (E) MCF-7 cells were treated for 16 h with either HRG (10 ng/ml) or vehicle in the presence or absence of SC205346 (3 μM), and CXCR4 was determined by flow cytometry. (Left) Representative dot plots for CXCR4 surface expression. (Middle) Percentages of CXCR4-positive cells. (Right) MFI for CXCR4 expression. The results are expressed as means and SEM of the results of 3 individual experiments. (F) MCF-7 cells were subjected to either HIF-1α or NTC RNAi. After 48 h, the cells were treated for 16 h with either HRG (10 ng/ml) or vehicle. The cell lysates were subjected to Western blotting with the indicated antibodies. (Left) Representative experiment. (Right) Densitometric analysis of HIF-1α expression, expressed as percentages relative to cells with HRG treatment. (G) Effect of HIF-1α RNAi on CXCR4 surface expression. MCF-7 cells were treated for 16 h with either HRG (10 ng/ml) or vehicle, and CXCR4 was determined by flow cytometry. (Left) Representative dot plots for CXCR4 surface expression. (Middle) Percentages of CXCR4-positive cells. (Right) Mean fluorescence intensities for CXCR4 expression. The results are expressed as means and SEM of 3 individual experiments. **, P < 0.01; ***, P < 0.001.

Article Snippet: The HIF-1α inhibitor SC205346 was obtained from EMD/Calbiochem (Gibbstown, NJ).

Techniques: Incubation, Western Blot, Positive Control, Expressing, Flow Cytometry, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α

doi: 10.1128/MCB.00180-16

Figure Lengend Snippet: HIF-1α mediates HRG-induced sensitization of Rac1 activation and motility. (A) MCF-7 cells were incubated for 16 h with either HRG (10 ng/ml) or vehicle (control) in the presence of the HIF-1α inhibitor SC205346 (3 μM, added 1 h before and during HRG or vehicle incubation). Four hours after HRG removal, the cells were treated with SDF-1 (100 ng/ml; 2 min), and Rac1-GTP levels were determined using a pulldown assay. (B) Rac1 activation by SDF-1 was determined in MCF-7 cells transfected with either HIF-1α or nontarget control RNAi duplexes. (A and B) (Left) Representative experiments. (Right) Densitometric values of Rac1-GTP levels (means and SEM, normalized to total Rac1; n = 3), expressed as fold increase relative to vehicle-treated NTC cells (minus SDF-1). (C) MCF-7 cells subjected to either HIF-1α or NTC RNAi were treated for 16 h with either HRG (10 ng/ml) or vehicle. Motility in response to SDF-1 (100 ng/ml; 16 h) was determined in a Boyden chamber. Shown is the effect of HIF-1α RNAi on MCF-7 cell motility. (Left) Representative images. (Right) Quantification of migrating cells by contrast microscopy in 5 independent fields. The results are expressed as means and SD of triplicate measurements. Two additional experiments gave similar results. C, control (vehicle). **, P < 0.01; ***, P < 0.001.

Article Snippet: The HIF-1α inhibitor SC205346 was obtained from EMD/Calbiochem (Gibbstown, NJ).

Techniques: Activation Assay, Incubation, Transfection, Microscopy

Journal: Molecular and Cellular Biology

Article Title: Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α

doi: 10.1128/MCB.00180-16

Figure Lengend Snippet: ErbB3 mediates HRG-induced sensitization of the CXCR4/Rac1 pathway in breast cancer cells. (A) MCF-7 cells were subjected to ErbB3, ErbB4, HIF-1α, or nontarget control RNAi. After 48 h, the cells were incubated for 6 h with either HRG (10 ng/ml) or vehicle (control). The cell lysates were subjected to Western blotting with the indicated antibodies. (Left) Representative experiment. (Right) Densitometric values of HIF-1α expression levels (means and SEM; n = 3) expressed as fold increase relative to cells without HRG treatment. (B) MCF-7 cells subjected to ErbB3, ErbB4, or NTC RNAi were treated for 16 h with either HRG (10 ng/ml) or vehicle (control). CXCR4 expression was determined by flow cytometry. (Left) Representative dot plots for CXCR4 surface expression. (Middle) Percentages of CXCR4-positive cells. (Right) Mean fluorescence intensities for CXCR4 expression. The results are expressed as means and SEM of the results of 3 individual experiments. (C) Effect of ErbB3 or ErbB4 RNAi on Rac1 activation by SDF-1 (100 ng/ml; 2 min) in MCF-7 cells treated with HRG or vehicle. (Left) Representative experiment. (Right) Densitometric values of Rac1-GTP levels (means and SEM, normalized to total Rac1; n = 3) expressed as fold increase relative to control cells, NTC (minus SDF-1). (D) Effect of ErbB3 or ErbB4 RNAi on MCF-7 cell motility in response to SDF-1 (0 to 100 ng/ml; 16 h) as determined with a Boyden chamber. The cells were previously treated for 16 h with either HRG (10 ng/ml) or vehicle (control). (Left) Representative images. (Right) Quantification of migrating cells by contrast microscopy in 5 independent fields. The results are expressed as means and SD of triplicate measurements. Two additional experiments gave similar results. **, P < 0.01; ***, P < 0.001; n.s., not significant.

Article Snippet: The HIF-1α inhibitor SC205346 was obtained from EMD/Calbiochem (Gibbstown, NJ).

Techniques: Incubation, Western Blot, Expressing, Flow Cytometry, Fluorescence, Activation Assay, Microscopy

Journal: Molecular and Cellular Biology

Article Title: Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α

doi: 10.1128/MCB.00180-16

Figure Lengend Snippet: Transcriptional activation of the human CXCR4 promoter by HRG is mediated by HIF-1α. (A) MCF-7 cells were treated with HRG (10 ng/ml) for the indicated times, fixed, and subjected to HIF-1α immunocytochemistry. The cells were counterstained with DAPI. (Right) Representative experiment. (Left) Quantification of cells with nuclear HIF-1α staining. (B) MCF-7 cells subjected to either HIF-1α or NTC RNAi were cotransfected with the pGL3-CXCR4 promoter construct and the Renilla luciferase expression vector pRL-TK (for normalization). The cells were treated for 12 h with either HRG (10 ng/ml) or vehicle, and luciferase activity was determined. A Western blot for HIF-1α is shown. (C) Luciferase reporter activities of mutated pGL3-CXCR4 promoter constructs. HRE-1, -2, and -3 sites are indicated with ovals. Mutated sites are marked with an X. (B and C) The data are expressed as means and SEM of three independent experiments. (D) ChIP assay for the HRE-1 site in the CXCR4 promoter in MCF-7 cells. The cells were treated for 12 h with either HRG (10 ng/ml) or vehicle. As a positive control, we used a region encompassing an HRE present in the VEGFA promoter. The ACTB promoter was used as a negative control. The sizes of the expected bands are indicated in each case. (Left) Representative ChIP assay. (Right) Densitometric analysis of HIF-1α binding. The data are presented as fold enrichment (HRG/vehicle) and expressed as means and SEM of the results of 3 independent experiments. **, P < 0.01; ***, P < 0.001.

Article Snippet: The HIF-1α inhibitor SC205346 was obtained from EMD/Calbiochem (Gibbstown, NJ).

Techniques: Activation Assay, Immunocytochemistry, Staining, Construct, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Western Blot, Positive Control, Negative Control, Binding Assay