Figure 1.

Screening for drugs that limit the muscle mononuclear cell adipogenic potential. (A) Flow diagram of the screening strategy. Muscle mononuclear cells were isolated from the hind limbs of wild type mice and seeded at a density of 4.5 × 10⁴ cells/cm² in matrigel coated well. Six-day cultured cells were incubated for 48 hours with adipogenic induction medium (ADM) supplemented with the Prestwick Chemical library® (PCL) compounds. Two days later, drug supplemented media were replaced by drug-free adipogenic maintenance medium (AMM) and cells were cultured for three additional days prior to staining with ORO solution. (B) Multiwell screening layout. For the control samples, cells were incubated in 0.5% (v/v) DMSO (green wells). Samples treated with 1 µM rosiglitazone (Rosi) were used as positive controls (red wells). Each compound was tested in a dose-response assay (1, 10, 25 μM) (white wells). Wells at the plate corners just contained medium and were used as negative controls (dashed wells). (C) Representative images, at 20x magnification, of Oil Red O (ORO)-staining of the primary screening. Muscle mononuclear cells were incubated in ADM with increasing concentration of azathioprine (AZA) (10, 25 μM) for 48 hours. Cells were then incubated for three additional days in AMM allowing adipocyte differentiation. (D,E) The floating bar graphs represent the average percentage of ORO-positive cells per field (D) and the average number of nuclei per well in each experimental condition (E). The statistical significance was estimated by one way ANOVA and defined as *p < 0.05; **p < 0.01; ***p < 0.001 (Vehicle n = 9, 1 μM AZA n = 2, 10 μM AZA n = 2, 25 μM AZA n = 2, 1 μM Rosi n = 10). Scale bar: (C) 100 μm.