Journal: bioRxiv

Article Title: Niacin fine-tunes energy homeostasis through canonical GPR109A signaling

doi: 10.1101/382416

Figure Lengend Snippet: A Phosphorylated of ERK1/2, AKT and AMPK were measured in the liver, muscle, and eWAT from the WT mice by western blot and quantitative analyses (n=5). B Phosphorylated of ERK1/2, AKT and AMPK were measured in the liver from Gpr109a −/− mice. (n=5). C Activation of ERK1/2, AKT, AMPK and ACC in serum-starved HepG2 cells stimulated with niacin (300 μM) for the indicated time periods. D HepG2 cells were transfected with GPR109A siRNA for 36 h and stimulated with niacin (300 μM) for 5 min. Phosphorylation of ERK1/2, AKT, AMPK and ACC was accessed. E Activation of ERK1/2, AKT, AMPK, and ACC in serum-starved HepG2 cells pretreated with PTX (100 ng/mL) for 16 hr or M119K (10 μM), Go6983 (10 μM), or U0126 (1 μM) for 1h followed by a challenge with niacin (300 μM) for 5 min was accessed by western blot. And β-tubulin was used as a loading control. F Schematic model of suppression of ACC via GPR109A. Data information: All the data are presented as means ±SEM. In (C,D,E), All data of cells shown are representative of at least three independent experiments. In (A,B), data were analyzed by unpaired two-tailed student’s t-test. * P < 0.05, ** P <0.01.

Article Snippet: Membranes were blocked with 5% BSA in TBS containing 0.1% Tween-20 (TBST) for 1 h at room temperature, and incubated overnight at 4°C with primary antibodies against p-ERK1/2 (Cat# 4370), ERK1/2 (Cat# 9102), p-AKT (Ser473) (Cat# 4058), AKT (Cat# 4691), p-AMPKα (Thr 172) (Cat# 2535), AMPKα (Cat# 5832), p-ACC (Ser79) (Cat# 11818), ACC (Cat# 3676), β-tubulin (Cat# 2128) (Cell Signaling Technology, USA), GPR109A (Santa Cruz Biotechnology, USA, Cat# sc-134583) respectively.

Techniques: Western Blot, Activation Assay, Transfection, Two Tailed Test

Journal: Frontiers in Physiology

Article Title: Odorant Receptor 51E2 Agonist β-ionone Regulates RPE Cell Migration and Proliferation

doi: 10.3389/fphys.2017.00888

Figure Lengend Snippet: β-ionone promotes proliferation and migration of RPE cells. (A) A gap closure assay, using RPE cells, in the presence of 10 μM, 100 μM or 0.1% DMSO (control). The open gap area after 12, 24, and 36 h is shown relative to the original gap area at time point 0 h. The 100 μM β-ionone treatment significantly enhanced cell migration compared to the control condition (0.1% DMSO) (right and left panel). (B) The proliferation of the RPE cells after treatment with increasing concentrations (1 μM, 5 μM, 10 μM, 50 μM, and 100 μM) of β-ionone for 5 days compared to the control condition. The relative cell number was determined by the CyQUANT cell proliferation assay. The data are shown as the mean of four independent experiments ± the SEM with technical replicates and were normalized to the cell number in the control experiments. (C) Immunofluorescence confocal micrographs of the RPE cells labeled with a PCNA-specific antibody (green) and AlexaFluor 546 phalloidin (red) (left panel). Immunocytochemical staining reveals significantly enhanced numbers of proliferating cells after stimulation with β-ionone (10 μM and 100 μM) compared to the control condition (0.1% DMSO). The data are shown as the mean of three independent experiments using 100 quantified cells for each experiment, which was normalized to the cell number in the control experiments. (D) The OR51E2 agonist β-ionone induces the phosphorylation of protein kinases in RPE cells. The Human Phospho-Kinase Array was plotted with proteins from the RPE cells stimulated with β-ionone (500 μM; lower panel) or control (0.1% DMSO; upper panel) for 10 min. The specific antibodies against the phosphorylated protein kinases were spotted in duplicate. The colored boxes mark areas where at least a 2-fold increase in the protein signal intensities between the β-ionone-treated cells and the solvent-treated cells (control) was observed. The pixel intensities of the duplicates were averaged and β-ionone-induced phosphorylation was normalized to the control. The phosphorylation of ERK1/2, AKT and PRAS40 was enhanced relative to the control (right panel; n = 1). (E) A Western blot analysis verified the phosphorylation of ERK1/2 (T202/Y204, T185/Y187) and AKT kinases (S473) in the RPE cells after 10 and 30 min stimulations with β-ionone (500 μM) compared to stimulation with the solvent (0.1% DMSO; control) (right panel). Determination of the total amounts of the respective kinases served as controls. The mean pixel intensities of the phosphorylated proteins relative to the total protein were quantified and normalized against the control-treated cells ( n = 4). Significance was calculated by Student's t -test ( * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.005).

Article Snippet: The following primary antibodies were used: custom-made rabbit polyclonal antibody against OR51E2 (Eurogentec; epitope: ISCDKDLQAVGGK); mouse monoclonal antibody against glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH; cat. no. #ab9485; Abcam); rabbit monoclonal antibody against PCNA (cat. no. #ab18197; Abcam); polyclonal rabbit anti-Gα s/olf antibody (cat. no. #sc-383; Santa Cruz Biotechnology, Dallas, Texas; USA), polyclonal rabbit anti-adenylyl cyclase III antibody (cat. no. #sc-588; Santa Cruz Biotechnology); rabbit monoclonal antibody against phospho-AKT (cat. no. #4060), AKT (cat. no. #4691), phospho-ERK1/2 (cat. no. #4370) and ERK1/2 (cat. no. #4695) (Cell Signaling Technology, Danvers, Massachusetts, USA); secondary goat-anti-rabbit and goat-anti-mouse antibodies conjugated to Alexa Fluor 546 or Alexa Fluor 488 (Life Technologies).

Techniques: Migration, CyQUANT Assay, Proliferation Assay, Immunofluorescence, Labeling, Staining, Western Blot