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

Journal: iScience

Article Title: Long-term intermittent hypoxia in mice induces inflammatory pathways implicated in sleep apnea and steatohepatitis in humans

doi: 10.1016/j.isci.2024.108837

Figure Lengend Snippet:

Article Snippet: Antibodies used for Western blots are as follows: anti-HIF1a (Abcam, ab2185), anti-HIF1a (Santa Cruz Biotechnologies, sc13515), anti-α-TUBULIN (Sigma, T5168), anti-Rabbit HRP (Jackson laboratories, 111-035-003), anti-Mouse HRP (Jackson laboratories, 115-035-003).

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Reverse Transcription, Avidin-Biotin Assay, Gene Expression, Software, Gas Chromatography-Mass Spectrometry, Microscopy, Luminex

Journal: PLoS ONE

Article Title: Orexin A affects HepG2 human hepatocellular carcinoma cells glucose metabolism via HIF-1α-dependent and -independent mechanism

doi: 10.1371/journal.pone.0184213

Figure Lengend Snippet: (A) HepG2 cells were treated with the indicated concentrations of orexin A for 2 h. HIF-1α mRNA expression was measured by real-time PCR. (B) HepG2 cells were treated with 10 −7 M orexin A for the indicated times. HIF-1α protein expression was measured by western blot. (C) HepG2 cells were treated with the indicated concentrations of orexin A for 2 h with or without the OX1R inhibitor, SB334867 (10 −6 M), HIF-1α inhibitor, YC-1 (10 −5 M), or a combination of both inhibitors. HIF-1α protein expression was measured by western blot. Data are presented as mean ± standard error of the mean based on analysis in triplicate. * P < 0.05 compared to control. (D) HepG2 cells were treated without or with 10 −7 M orexin A under normoxia for 2 h, or 150 uM CoCl 2 (stimulating hypoxia) for 8 h. The cellular localization of HIF-1α was measured by immunofluorescent staining using the anti-HIF-1α antibody. Nucleus was immunolabeled with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI). Staining was analyzed by fluorescence microscope.

Article Snippet: Cobalt chloride (CoCl 2 ), Akt inhibitor (LY294002), mTOR inhibitor (temsirolimus) and HIF-1α inhibitor (YC-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Immunolabeling, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Orexin A affects HepG2 human hepatocellular carcinoma cells glucose metabolism via HIF-1α-dependent and -independent mechanism

doi: 10.1371/journal.pone.0184213

Figure Lengend Snippet: HepG2 cells were treated with or without 10 −7 M orexin A for 30 min in the presence or absence of 10 −5 M LY294002 (Akt inhibitor) or 10 −5 M temsirolimus (mTOR inhibitor), 10 −6 M SB334867 (OX1R inhibitor), as well as the combination of both inhibitors. The phosphorylation of Akt (A) and mTOR (B) was normalized against the total protein expression. The total protein expression was used as an internal control for equal protein loading. Protein activation was measured by western blot. (C) Cells were treated with or without 10 −7 M orexin A for 2 h in the presence or absence of 10 −5 M LY294002 (Akt inhibitor), 10 −5 M temsirolimus (mTOR inhibitor), or the combination of both inhibitors. HIF-1α protein expression was measured by western blot. Data are presented as mean ± standard error of the mean based on experimental analysis in triplicate. * P < 0.05 compared to control.

Article Snippet: Cobalt chloride (CoCl 2 ), Akt inhibitor (LY294002), mTOR inhibitor (temsirolimus) and HIF-1α inhibitor (YC-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Activation Assay, Western Blot

Journal: PLoS ONE

Article Title: Orexin A affects HepG2 human hepatocellular carcinoma cells glucose metabolism via HIF-1α-dependent and -independent mechanism

doi: 10.1371/journal.pone.0184213

Figure Lengend Snippet: Cells were treated with the indicated doses of orexin A with or without the OX1R inhibitor SB334867 (10 −6 M), HIF-1α inhibitor, YC-1 (10 −5 M), or a combination of both inhibitors. (A) After 2 h, GLUT1 protein expression were determined by western blot. (B) After 30 min, glucose uptake was also measured. Data are presented as mean ± standard error of the mean based on experimental analysis in triplicate. * P < 0.05 compared to control.

Article Snippet: Cobalt chloride (CoCl 2 ), Akt inhibitor (LY294002), mTOR inhibitor (temsirolimus) and HIF-1α inhibitor (YC-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Orexin A affects HepG2 human hepatocellular carcinoma cells glucose metabolism via HIF-1α-dependent and -independent mechanism

doi: 10.1371/journal.pone.0184213

Figure Lengend Snippet: Cells were treated with the indicated doses of orexin A for 2 h with or without the OX1R inhibitor SB334867 (10 −6 M), HIF-1α inhibitor, YC-1 (10 −5 M), or a combination of both inhibitors. ATP content was determined using an ATP Assay kit. Data are presented as mean ± standard error of the mean based on experimental analysis in triplicate. * P < 0.05, ** P < 0.01 compared to control.

Article Snippet: Cobalt chloride (CoCl 2 ), Akt inhibitor (LY294002), mTOR inhibitor (temsirolimus) and HIF-1α inhibitor (YC-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: ATP Assay

Journal: PLoS ONE

Article Title: Orexin A affects HepG2 human hepatocellular carcinoma cells glucose metabolism via HIF-1α-dependent and -independent mechanism

doi: 10.1371/journal.pone.0184213

Figure Lengend Snippet: Cells were treated with or without 10 −7 M orexin A for the indicated times in the presence or absence of the OX1R inhibitor SB334867 (10 −6 M) and HIF-1α inhibitor, YC-1 (10 −5 M). (A) After 2 h, PDHB, LDHA, PDK1 mRNA expression were determined by real-time PCR. (B) Lactate generation was measured using a Lactic Acid assay kit. (C) PDH enzyme activity was determined using a Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit. Data are presented as mean ± standard error of the mean based on experimental analysis in triplicate. * P < 0.05, ** P < 0.01 compared to control.

Article Snippet: Cobalt chloride (CoCl 2 ), Akt inhibitor (LY294002), mTOR inhibitor (temsirolimus) and HIF-1α inhibitor (YC-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Acid Assay, Activity Assay