FIG. 2.

BMP7 treatment rescued brown adipocyte differentiation of IRS-1KO cells. (A) Differentiation protocol. IRS-1KO cells were pretreated with vehicle or BMP7 for 3 days and then induced to differentiate, as described in Materials and Methods. As a control, WT cells were treated with vehicle only and then induced to differentiate. RNA and protein were isolated on day 3 and day 10 and analyzed by Q-RT-PCR and Western blotting, respectively. On day 10, the cells were fixed and stained with Oil Red O. (B) Oil Red O staining of cells at day 10. Pre-Tx, pretreatment. (C) Q-RT-PCR analysis of the expression of adipogenic markers at day 10. (D) Q-RT-PCR analysis of the expression of brown-fat-selective genes at day 10. V, vehicle. For Q-RT-PCR analysis, data are presented as the mean plus the standard error of the mean (SEM) from a representative of 4 independent experiments, with each performed in triplicate. The values for WT cells were set as 100 arbitrarily. Statistically significant differences between vehicle- and BMP7-treated IRS-1KO cells are indicated above the bars: *, P < 0.05; **, P < 0.01; ***, P < 0.0001. (E) Western blotting of general adipogenic and brown-fat-specific marker proteins. A representative of 4 independent experiments is shown.