Journal: Journal of Biological Chemistry

Article Title: Constitutively Active Homo-oligomeric Angiotensin II Type 2 Receptor Induces Cell Signaling Independent of Receptor Conformation and Ligand Stimulation

doi: 10.1074/jbc.m500639200

Figure Lengend Snippet: FIG. 1. a, AT2 receptors form homo-oligomerization and up-regulate after serum-free conditions in PC12W cells. Cell membrane was prepared under serum or serum-free conditions, subjected to SDS-gel electrophoresis under non-reducing conditions, and immunoblotted with anti-AT2 receptor antibody. 30 g of protein was used in each lane. Bmax on the AT2 receptors was also analyzed by Scatchard plot analysis. b, AT2-WT-EGFP receptor localized in the cell membrane after 24 h under serum-free conditions. EGFP- and AT2-WT-EGFP receptor-expressing CHO cell lines were grown under serum or serum-free conditions and stained with DAPI to visualize nuclear morphology as assessed by a laser scanning confocal microscope. Arrows indicate AT2-WT-EGFP receptor translocation in the cell membrane. c, AT2-WT-EGFP receptor induced apoptosis after 48 h under serum-free conditions. AT2-WT-EGFP receptor- and AT2-N127G-EGFP receptor-expressing CHO cell lines were grown under serum conditions or for up to 48 h under serum-free conditions, stained with DAPI, and imaged by a digital fluorescent microscope. d, pharmacological intervention in apoptosis mediated by AT2-WT-EGFP receptor and AT2-N127G-EGFP receptor was analyzed by treating cells under serum conditions and with or without 10 M p38 MAPK inhibitor (SB203580) and 1 M caspase-3 inhibitor (DEVD-cmk) for 48 h under serum-free conditions. Data are shown as the percentage of apoptotic cells in three independent experiments as assessed by TUNEL as described under “Experimental Procedures.” Histograms show that AT2-WT-EGFP receptor transfected CHO cells induced apoptosis under serum (A) or after 48 h serum-free conditions (B). 65% of AT2-WT-EGFP receptor transfected CHO cells showed apoptosis after 48 h of serum-free conditions. *, p 0.05 versus serum conditions. t, p 0.05 versus serum-free conditions without treatment. Data are given as mean S.E. (n 4). e, the AT2 receptor formed a homo-oligomer in the cell membrane. Cell membranes were prepared in serum-free conditions for 24 h, subjected to SDS-gel electro- phoresis after being pretreated with () or without ( ) DTT under non-reducing conditions, and immunoblotted with anti-EGFP antibody. 20 mg of protein was used in each lane. Arrows indicate the monomer and dimer species of the receptor. f, AT2-WT-EGFP receptor formed only homo-oligomer, whereas AT2-N127G-EGFP receptor formed both homo-oligomer and monomer in the cell membrane. Cell membranes were prepared in serum () or serum-free ( ) conditions for up to 48 h, subjected to SDS-gel electrophoresis under non-reducing conditions, and immunoblotted with anti-EGFP antibody. 40 g of protein was used in each lane. Arrows indicate the monomer and dimer species of the receptor. Representative immunoblots (a, d, and f) and pictures (b and c) are shown. Three independent determinations were performed, and similar results were observed (a–d and f).

Article Snippet: Materials—The following antibodies and reagents were generously provided as indicated or purchased: AT2 receptor-selective non-peptide antagonist PD123319 (Research Biochemical International) and the p38 MAP kinase inhibitor SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)1H-imidazole (Upstate Biotechnology); the caspase-3 inhibitor Ac-DEVD-chloro methyl ketone (Calbiochem); antiAT2 receptor antibody catalog number sc-7420 (Santa Cruz Biotechnology).

Techniques: Membrane, SDS-Gel, Electrophoresis, Expressing, Staining, Microscopy, Translocation Assay, TUNEL Assay, Transfection, Western Blot

Journal: International Journal of Endocrinology

Article Title: Effect of Gelam Honey on the Oxidative Stress-Induced Signaling Pathways in Pancreatic Hamster Cells

doi: 10.1155/2013/367312

Figure Lengend Snippet: Effect of quercetin and Gelam honey extract on pAkt (Ser473) expression.Quantitative analysis and representative western blot analysis of pAkt (Ser473) in HIT-T15 cells pretreated with quercetin and honey extract in cells cultured in 20 mM ((a), (c), (e)) and 50 mM ((b), (d), (f)) glucose. A sustained increase in the level of pAkt (ser473) was observed after pretreatment with quercetin and honey extract. Akt inhibitor VIII prevented the expression Akt ser473 phosphorylation induced by quercetin and honey extract. The results were normalized with β actin antibody. Data were presented as the mean ± standard deviation. (e) * P < 0.05; # P < 0.005 quercetin and honey extract treated compared to the 20 mM glucose alone. (f) * P < 0.05; # P < 0.005 quercetin and honey extract treated compared to the 50 mM glucose alone.

Article Snippet: To investigate inhibitory effects on Akt signaling pathway, cells were incubated with 5 μ M Akt inhibitor VIII (Santa Cruz Biotechnology sc-203173) for one hour before pretreatment with quercetin and honey extract.

Techniques: Expressing, Western Blot, Cell Culture, Phospho-proteomics, Standard Deviation

Journal: International Journal of Endocrinology

Article Title: Effect of Gelam Honey on the Oxidative Stress-Induced Signaling Pathways in Pancreatic Hamster Cells

doi: 10.1155/2013/367312

Figure Lengend Snippet: The effect of flavonoids and Gelam honey extract on insulin content. (a) Effect of pretreatment with quercetin and Gelam honey extract and the addition of Akt inhibitor VIII on the insulin content in cells cultured in 20 mM glucose. There was a significant increase in insulin content (* P < 0.05) when the cells were pretreated with quercetin and honey. There was a significant decrease in insulin content (* P < 0.05) when the cells were treated with Akt inhibitor VIII, before pretreating with quercetin and Gelam honey extract. (b) Effect of pretreatment with quercetin and Gelam honey and the addition of Akt inhibitor VIII on the insulin content in cells cultured in 50 mM glucose. There was a significant increase in insulin content (* P < 0.05, # P < 0.005) when the cells were pretreated with quercetin and honey. There was a significant decrease in insulin content (* P < 0.05, # P < 0.005) when the cells were treated with Akt inhibitor VIII, before pretreating with quercetin and Gelam honey extract.

Article Snippet: To investigate inhibitory effects on Akt signaling pathway, cells were incubated with 5 μ M Akt inhibitor VIII (Santa Cruz Biotechnology sc-203173) for one hour before pretreatment with quercetin and honey extract.

Techniques: Cell Culture

Journal: International journal of molecular sciences

Article Title: Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts.

doi: 10.3390/ijms23169086

Figure Lengend Snippet: Figure 4. Involvement of p38 MAPK in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 3 µM p38 inhibitor VIII (p38i VIII) for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 3 µM p38i VIII for 1 h and then incubated with 5 ng/mL TNF-α for the indicated time. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or p38α siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of p38α were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 10 µM AG1478 or 3 µM p38i VIII for 1 h and then treated with TNF-α for the indicated times (0, 5, 10, 15, 30, and 60 min). The phosphorylation of p38 and EGFR was determined by Western blot with GAPDH as a loading control. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). # p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Article Snippet: Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay, Transfection, Western Blot, Control, Phospho-proteomics

Journal: International journal of molecular sciences

Article Title: Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts.

doi: 10.3390/ijms23169086

Figure Lengend Snippet: Figure 6. Involvement of FoxO1 in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 1 µM AS1842856 for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 1 µM AS1842856 for 1 h and then incubated with 5 ng/mL TNF-α for the indicated time. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or FoxO1 siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of FoxO1 were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 1 µM AS1842856, 3 µM p38i VIII, or 3 µM SP600125 for 1 h and then treated with TNF-α for the indicated times (0, 5, 10, 15, 30, and 60 min). The phosphorylation of FoxO1, p38, or JNK1/2 was determined by Western blot with GAPDH as a loading control. (E) Cells were pretreated without or with 3 µM p38i VIII, 3 µM SP600125, or 1 µM AS1842856 for 1 h and then stimulated with TNF-α for the indicated time intervals or 30 min. The binding of FoxO1 to the promoter region of CCL20 was determined with a ChIP assay. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). # p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Article Snippet: Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay, Transfection, Western Blot, Control, Phospho-proteomics, Binding Assay

Journal: International journal of molecular sciences

Article Title: Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts.

doi: 10.3390/ijms23169086

Figure Lengend Snippet: Figure 8. Schematic diagram illustrating the proposed signaling pathway involved in TNF-α-induced CCL20 expression and secretion in HCFs. TNF-α-induced CCL20 expression was, at least partially, mediated through binding to TNFR1 leading to transactivation of EGFR. Activated EGFR promoted the phosphorylation of p38 MAPK- or JNK1/2-dependent FoxO1 activation, which further bound with the FoxO1 response element (FRE) on the CCL20 promoter. In addition, TNF-α also turned on NF-κB transcription factors. Either FoxO1 or NF-κB activation could enhance the expression of CCL20 induced by TNF-α, which may be engaged in the inflammatory responses in HCFs. A better understanding of mechanisms underlying the regulation of the CCL20 gene by TNF-α will support more opportunities to develop anti-inflammatory therapeutic strategies for treating cardiac inflammation.

Article Snippet: Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Binding Assay, Phospho-proteomics, Activation Assay