Journal: International Journal of Endocrinology

Article Title: Interaction between Angiotensin II and Insulin/IGF-1 Exerted a Synergistic Stimulatory Effect on ERK1/2 Activation in Adrenocortical Carcinoma H295R Cells

doi: 10.1155/2016/3403292

Figure Lengend Snippet: Ang II caused dose-dependent activation of ERK1/2 in H295R cells. Serum-starved H295R cells were treated with Ang II at different doses (1, 10, 100, and 1000 nM) for 5 min, then lysed in Laemmli sample buffer, and analyzed by SDS-PAGE using antibody against phospho-ERK1/2 (Thr202/Tyr204) and phospho-AKT (Ser473). The bolt was reprobed with total ERK1/2 and total AKT antibody loading controls.

Article Snippet: Human recombinant Ang II, insulin, IGF-1, and AT2 receptor blocker PD123319 were procured from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Activation Assay, SDS Page

Journal: International Journal of Endocrinology

Article Title: Interaction between Angiotensin II and Insulin/IGF-1 Exerted a Synergistic Stimulatory Effect on ERK1/2 Activation in Adrenocortical Carcinoma H295R Cells

doi: 10.1155/2016/3403292

Figure Lengend Snippet: Effect of Ang II and insulin/IGF-1, alone or combined, on phosphorylation of ERK1/2 and AKT in H295R cells. (a-b) Serum-starved H295R cells were treated with increasing concentrations of insulin/IGF-1 for 5 min with or without 30-minute preincubation with 100 nM Ang II. (a) Insulin (10, 100, and 1000 nM); (b) IGF-1 (0.1, 1, 10 nM). (c-d) Serum-starved H295R cells were treated with insulin (100 nM)/IGF-1 (10 nM) for 5 min with or without 30-minute preincubation with 100 nM Ang II. The histogram shows the densitometric result of the phosphorylation of three independent experiments. ERK1/2 and AKT phosphorylation in control group were taken as 100%. ∗ P < 0.05 versus control, ∗∗ P < 0.01 versus control, ## P < 0.01 versus Ang II alone, and %% P < 0.01 versus insulin/IGF-1 alone.

Article Snippet: Human recombinant Ang II, insulin, IGF-1, and AT2 receptor blocker PD123319 were procured from Sigma-Aldrich (St. Louis, MO, USA).

Techniques:

Journal: International Journal of Endocrinology

Article Title: Interaction between Angiotensin II and Insulin/IGF-1 Exerted a Synergistic Stimulatory Effect on ERK1/2 Activation in Adrenocortical Carcinoma H295R Cells

doi: 10.1155/2016/3403292

Figure Lengend Snippet: Effects of blockage of AT1R, AT2R, and IGF-1R on phosphorylation of ERK1/2 and AKT in H295R cells. H295R cells were treated with candesartan (1 μ M), PD1233319 (1 μ M), and MAB 391 (20 μ g/mL) for 30 min, then incubated with Ang II (100 nM) for 30 min, and then stimulated with insulin/IGF-1 for 5 min. (a) Insulin (100 nM); (b) IGF-1 (10 nM).

Article Snippet: Human recombinant Ang II, insulin, IGF-1, and AT2 receptor blocker PD123319 were procured from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Incubation

Journal: International Journal of Endocrinology

Article Title: Interaction between Angiotensin II and Insulin/IGF-1 Exerted a Synergistic Stimulatory Effect on ERK1/2 Activation in Adrenocortical Carcinoma H295R Cells

doi: 10.1155/2016/3403292

Figure Lengend Snippet: Involvement of MEK in agonists-stimulated phospho-ERK1/2 and phospho-AKT in H295R cells. (a) Effects of U0126, a MEK1/2 inhibitor, on Ang II-induced ERK1/2 and AKT phosphorylation. H295R cells were treated with increasing concentrations of U0126 for 30 min and then stimulated with Ang II (100 nM) for 5 min. (b) Quantitative determination of the effects of U0126 on ERK1/2 and AKT phosphorylation by Ang II from panels (a). (c and d) After incubation with U0126 (10 μ M) for 30 min, H295R cells were pretreated with Ang II for another 30 min and then stimulated with insulin/IGF-1 for 5 min. (c) Insulin (100 nM); (d) IGF-1 (10 nM).

Article Snippet: Human recombinant Ang II, insulin, IGF-1, and AT2 receptor blocker PD123319 were procured from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Incubation

Journal: International Journal of Endocrinology

Article Title: Interaction between Angiotensin II and Insulin/IGF-1 Exerted a Synergistic Stimulatory Effect on ERK1/2 Activation in Adrenocortical Carcinoma H295R Cells

doi: 10.1155/2016/3403292

Figure Lengend Snippet: Involvement of PKC in agonists-stimulated phospho-ERK1/2 and phospho-AKT in H295R cells. (a) Effects of Gö6983, a PKC inhibitor, on Ang II-induced ERK1/2 and AKT phosphorylation. H295R cells were treated with increasing concentrations of Gö6983 for 30 min and then stimulated with Ang II (100 nM) for 5 min. (b) Quantitative determination of the effects of Gö6983 on Ang II-induced ERK1/2 and AKT phosphorylation from panels (a). (c and d) After incubation with Gö6983 (5 μ M) for 30 min, H295R cells were pretreated with Ang for another 30 min and then stimulated with insulin/IGF-1 for 5 min. (c) Insulin (100 nM); (d) IGF-1 (10 nM).

Article Snippet: Human recombinant Ang II, insulin, IGF-1, and AT2 receptor blocker PD123319 were procured from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Incubation

Journal: International Journal of Endocrinology

Article Title: Interaction between Angiotensin II and Insulin/IGF-1 Exerted a Synergistic Stimulatory Effect on ERK1/2 Activation in Adrenocortical Carcinoma H295R Cells

doi: 10.1155/2016/3403292

Figure Lengend Snippet: Involvement of PI3K in agonists-stimulated phospho-ERK1/2 and phospho-AKT in H295R cells. (a) Effects of LY294002, a PI3K inhibitor, on insulin-induced ERK1/2 and AKT phosphorylation. H295R cells were treated with increasing concentrations of LY294002 for 30 min and then stimulated with insulin (100 nM) for 5 min. (b) Quantitative determination of the effects of LY294002 on insulin-induced ERK1/2 and AKT phosphorylation from panels (a). (c and d) After incubation with LY294002 (5 μ M) for 30 min, H295R cells were pretreated with Ang II (100 nM) for another 30 min and then stimulated with insulin/IGF-1 for 5 min. (c) Insulin (100 nM); (d) IGF-1 (10 nM).

Article Snippet: Human recombinant Ang II, insulin, IGF-1, and AT2 receptor blocker PD123319 were procured from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Incubation

Journal: International Journal of Endocrinology

Article Title: Interaction between Angiotensin II and Insulin/IGF-1 Exerted a Synergistic Stimulatory Effect on ERK1/2 Activation in Adrenocortical Carcinoma H295R Cells

doi: 10.1155/2016/3403292

Figure Lengend Snippet: Effect of interaction between Ang II and insulin/IGF-1 on the expression of steroidogenic enzymes. H295R cells were treated with Ang II (100 nM) and insulin (100 nM)/IGF-1 (10 nM), combined or alone, for 24 hours. mRNA expressions of CYP11B1 and CYP11B2 were detected. ∗∗ P < 0.01 versus control and # P < 0.05 versus Ang II.

Article Snippet: Human recombinant Ang II, insulin, IGF-1, and AT2 receptor blocker PD123319 were procured from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Clusterin/Apolipoprotein J Attenuates Angiotensin II-Induced Renal Fibrosis

doi: 10.1371/journal.pone.0105635

Figure Lengend Snippet: Representative images of renal cortex sections from wild-type (Clu +/+ ) and Clu -/- mice treated with or without Ang II for 14 d. ( A ) The sections were stained with H&E or Sirius red, or were immunostained with antibodies targeting PAI-1, fibronectin, and type I collagen. The number of atrophic tubules was determined by measurement of the abnormal irregular and dilated tubular basement membranes in five random fields of H&E-stained sections under high-power magnification. Areas of positive staining with Sirius red and positive immunostaining with PAI-1, fibronectin, and type I collagen antibodies were quantified by computer-based morphometric analysis. ( B ) The sections were immunostained with an antibody targeting AT1R and p-Smad3. Areas of positive immunostaining were quantified by computer-based morphometric analysis. Data were normalized to the untreated wild-type and are expressed as the mean ± SEM of n = 5 random fields of each kidney (n = 5 in each group). * P <0.01, # P <0.05, and † P <0.001 compared with untreated wild-type mice and ‡ P <0.01, § P <0.05, and || P <0.001 compared with Ang II-treated wild-type mice. Original magnification, ×200.

Article Snippet: The recombinant human Ang II was purchased from Sigma (St. Louis, MO).

Techniques: Staining, Immunostaining

Journal: PLoS ONE

Article Title: Clusterin/Apolipoprotein J Attenuates Angiotensin II-Induced Renal Fibrosis

doi: 10.1371/journal.pone.0105635

Figure Lengend Snippet: Expression levels of the indicated mRNAs and proteins in untreated and Ang II-stimulated NRK-52E cells. The cells were rendered quiescent by incubation for 24 h and then infected with the indicated doses of Ad-clusterin-GFP (Ad-Clusterin) or Ad-GFP for 2 h. After incubation for a further 20 h, the cells were incubated with 200 nM Ang II for 4 h. ( A and B ) Representative real-time RT-PCR ( A ) and immunoblot ( B ) analyses of the expression levels of PAI-1, type I collagen, and fibronectin. ( C and D ) Representative real-time RT-PCR of AT1R ( C ) and immunoblot ( D ) analyses of the expression of levels of AT1R and p-Smad3. The mRNA expression levels were normalized to those of GAPDH and the data are represented as the mean ± SEM of n = 3 independent measurements (n = 3 separate experiments). The protein expression levels were normalized to those of β-actin and data are expressed as the mean ± SEM of n = 3 independent experiments (n = 5 in each group). ¶ P <0.01, ** P<0.05 and ## P <0.001 compared with control (untreated cells); †† P <0.01, ‡‡ P <0.05, and §§ P <0.001 compared with Ang II alone.

Article Snippet: The recombinant human Ang II was purchased from Sigma (St. Louis, MO).

Techniques: Expressing, Incubation, Infection, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Clusterin/Apolipoprotein J Attenuates Angiotensin II-Induced Renal Fibrosis

doi: 10.1371/journal.pone.0105635

Figure Lengend Snippet: Representative images of renal sections from rats infected with Ad-GFP or Ad-Clusterin and treated with Ang II for 14 d. ( A ) The sections were stained with H&E or Sirius red, or were immunostained with antibodies targeting PAI-1, type I collagen, and fibronectin. The number of atrophic tubules was determined by measurement of the abnormal irregular and dilated tubular basement membranes in five random fields of H&E-stained sections under high-power magnification. Areas of positive staining with Sirius red and positive immunostaining with PAI-1, type I collagen, and fibronectin antibodies were quantified by computer-based morphometric analysis. ( B ) The sections were immunostained with an antibody targeting AT1R and p-Smad3. Areas of positive immunostaining were quantified by computer-based morphometric analysis. Data were normalized to the control and are represented as the mean ± SEM of five random fields of each kidney (n = 5 in each group). Original magnification, ×200. |||| P <0.01, ¶¶ P <0.05, and *** P <0.001 compared with the control; ### P <0.01, and ††† P <0.001 compared with Ad-GFP.

Article Snippet: The recombinant human Ang II was purchased from Sigma (St. Louis, MO).

Techniques: Infection, Staining, Immunostaining

Journal: PLoS ONE

Article Title: Clusterin/Apolipoprotein J Attenuates Angiotensin II-Induced Renal Fibrosis

doi: 10.1371/journal.pone.0105635

Figure Lengend Snippet: Rats were infected with Ad-GFP or Ad –clusterin and treated with Ang II for 14 d. (A and B) Representative real-time RT-PCR (A) and immunoblot (B) analyses of the renal expression levels of clusterin, PAI-1, type I collagen, and fibronectin. (C and D) Representative real-time RT-PCR of AT1R (C) and immunoblot (D) analyses of the renal expression levels of AT1R and p-Smad3. The mRNA expression levels were normalized to those of GAPDH and the protein expression levels were normalized to those of β-actin. The data are represented as the mean ± SEM of n = 3 independent measurements (n = 5 in each group). |||| P <0.01, ¶¶ P <0.05, and *** P <0.001 compared with the control; ### P <0.01 and ††† P <0.001 compared with Ad-GFP.

Article Snippet: The recombinant human Ang II was purchased from Sigma (St. Louis, MO).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Clusterin/Apolipoprotein J Attenuates Angiotensin II-Induced Renal Fibrosis

doi: 10.1371/journal.pone.0105635

Figure Lengend Snippet: NRK-52E cells were infected with Ad-clusterin (100 moi) or Ad-GFP (100 moi) as a control after a 24 h serum starvation. After incubation for a further 20 h, the cells were incubated with 200 nM Ang II for 8 h. (A) Representative immunoblot analysis of the expression of AT1R, p-IkBα, IkBα, p-NF-kB and clusterin in Ang II untreated or stimulated NRK-52E cells. β-actin was used as an internal control. ¶ P<0.01, ** P<0.05, and ## P<0.001 compared with control (untreated cells); †† P<0.01, ‡‡ P<0.05, and §§ P<0.001 compared with Ang II alone. (B) Representative immunoblot of the expression of p-NF-κB in untreated and Ang II-stimulated NRK-52E cells. Nuclear and cytoplasmic (Cyto) proteins were separated by SDS-PAGE and immunoblots were performed using anti-β-actin, anti-Lamin B, and anti-p-NF-κB antibodies. (C) Representative images of immunofluorescent detection of anti-p-NF-κB in untreated and Ang II-stimulated NRK-52E cells. The cellular localizations of p-NF-κB (red), clusterin (green) and nuclei stained with Hoechst (blue) were examined using fluorescence microscopy. Magnification, ×400. (D) Co-immunoprecipitation (IP) of clusterin and NF-κB in Ang II-treated NRK-52E cells. IP was performed using cell lysates and an anti-NF-κB antibody. Samples were analyzed by immunoblotting with anti-clusterin and anti- NF-κB antibodies. As a loading control, β-actin was detected in the input samples.

Article Snippet: The recombinant human Ang II was purchased from Sigma (St. Louis, MO).

Techniques: Infection, Incubation, Western Blot, Expressing, SDS Page, Staining, Fluorescence, Microscopy, Immunoprecipitation