Probes were

Probes were www.selleckchem.com/products/Enzastaurin.html labeled using 50 ��Ci [��-32P]ATP (MP Biomedicals, Solon, OH) and then purified on Quick Spin Sephadex G25 columns (Roche). Sequences for miRNA378, 378*, 199b, 103, and 21 probes are respectively the following: 5��-ggccttctgactccaagtccag-3��, 5��-acacaggacctggagtcaggag-3��, 5��-gaacagatagtctaaacactggg-3��, 5��-tcatagccctgtacaatgctgct-3��, and 5��-tcaacatcagtctgataagcta-3��. Membrane was prehybridized in rapid hybridization buffer (GE Healthcare Life Sciences, Piscataway, NJ) at 42��C. After denaturing the probe, the membrane was incubated overnight at 42��C in hybridization buffer and then washed three times for 7 min at 32��C in SSC 2��, SDS 0.1%. Quantification of Northern blots was performed by phosphorimaging using Quantity One Software (Bio-Rad). Triacylglycerol measurements.

The amount of cellular triacylglycerol was determined with Infinity triglyceride reagent (Sigma-Aldrich, St. Louis, MO) with glycerol as the standard. RESULTS Ago2 is required for efficient synthesis of triacylglycerols. Ago2 plays a key role in the processing and stability of miRNAs, as well as action of endogenous siRNAs; therefore, to investigate potential roles of miRNAs on aspects of adipocyte biology, we evaluated effects of Ago2 deficiency on adipocyte differentiation and metabolism (3). We stably knocked down expression of Ago2 in 3T3-L1 preadipocytes using a retroviral shRNA expression vector (shAgo2) (20). Ago2 mRNA was reduced by ~72% in Ago2 knock-down adipocytes at day 7 of differentiation (Fig. 1A). By phase-contrast microscopy, the rate and extent of adipogenesis did not appear to be different.

Similarly, adipocyte morphology including size and number of lipid droplets appear similar (Fig. 1B). To confirm and extend these observations, we quantified expression of several adipocyte genes and found that C/EBP�� mRNA levels are similar between control and shAgo2 3T3-L1 preadipocytes and adipocytes at day 8 (Fig. 1C). However, Western blot analysis revealed a slight but significant increase in expression of PPAR�� and C/EBP�� proteins in adipocytes with an Ago2 deficiency, although differences in FABP4 were not observed (Fig. 1D). Taken together, these data suggest that differentiation of adipocytes is not substantially influenced by knockdown of Ago2. Fig. 1. Ago2 is required for efficient incorporation of [14C]glucose into triacylglycerol.

A: knockdown of Ago2 in 3T3-L1 adipocytes. After retroviral transduction with shRNAs, Ago2 expression levels in day 7 3T3-L1 adipocytes were measured by quantitative RT-PCR, … To assess a potential Cilengitide role of Ago2 in lipid metabolism, we examined effects of Ago2 knock-down on lipogenesis. After metabolic labeling with [14C]acetate for 45 min or with [14C]glucose for 2 h, lipids were extracted and separated by thin-layer chromatography (Fig. 1, E and F, respectively).

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