Journal: Cells

Article Title: The Novel Inhibitory Effect of YM976 on Adipocyte Differentiation

doi: 10.3390/cells12020205

Figure Lengend Snippet: AMPK inhibitor lessens the inhibitory effect of YM976 on adipocyte differentiation. ( A – D ) Cells were pre-treated with Compound C (1 µM) for 1 h and then incubated with YM976 during adipocyte differentiation for six days. ( A ) Cells were stained with Oil Red O and observed by microscopy. Scale bar = 100 µm. ( B ) Quantification of Oil Red O staining by spectrophotometry. ( C ) The mRNA expression levels of PPARγ, C/EBPα, and FASN were examined by qRT-PCR. ( D ) Levels of indicated proteins were analyzed by immunoblotting. ( E ) After silencing with si-NS or si-AMPKα, 3T3-L1 cells were transfected with PPARγ expression plasmid and pGL3 luciferase vector (pGL3) or PPAR-response element-binding luciferase vector (PPRE-Luc) for 24 h. Cells were then treated with Veh or YM976 for 24 h. Luciferase assay was performed to measure the transcription activity of PPARγ. ( F ) After pre-incubation with Veh or Compound C (1 µM) for 1 h, 3T3-L1 cells were transfected with PPARγ expression plasmid and pGL3 or PPRE-Luc or for 24 h. Cells were then treated with Veh or YM976 for 24 h. Luciferase assay was performed to measure the transcriptional activity of PPARγ. ( G ) Proposed model of YM976 inhibition of adipocyte differentiation. YM976 increases the levels of cAMP and AMPK phosphorylation to suppress the transcriptional expression of adipogenic genes, resultings in reduced adipogenesis. pGL3, pGL3 luciferase vector; pPPRE, peroxisome proliferator response element reporter plasmid; C.C., Compound C; N.S., not significant. In ( B , C , E , F ), n = 3 per group. In A and D, images and blots are representative of three independent experiments. Values indicate mean ± SD. * p < 0.05; ** p < 0.01. Data were assessed by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Antibodies against phospho-AMPK (cat. 2535), AMPKα (cat. 2532), C/EBPα (cat. 2295), FASN (cat. 3189), FABP4 (cat. 2120), phospho-CREB (cat. 9198), CREB (cat. 9197), phosphor-ACC (cat. 3661), and ACC (cat. 3662) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Incubation, Staining, Microscopy, Spectrophotometry, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Luciferase, Binding Assay, Activity Assay, Inhibition