Supplementary Figure 2: Gain- and loss-of-function experiments showed the effect of NFIA on the brown fat gene program.
From: NFIA co-localizes with PPARγ and transcriptionally controls the brown fat gene program
(a) Hierarchal clustering analysis of genes up- or down-regulated by NFIA. For example, genes up-regulated NFIA (Cluster A) include Ucp1, Ppargc1a, and Adrb3—the vital components for the induction of thermogenesis in response to adrenergic stimulus, as well as Pparg—the master regulator of adipogenesis. In contrast, Cluster B represents genes down-regulated by NFIA, including critical myogenic genes such as Myog, Myh1, and Myl1. Note that the clusters of genes up-regulated by NFIA largely overlap with BAT-selective genes (p = 9.9 × 10−28), and the cluster of genes down-regulated by NFIA overlap with SKM-selective genes (p = 2.3 × 10−32). The definition of BAT- and SKM- selective genes (N = 254 and N = 312, respectively) are shown in the methods. The analyses were performed once based on the RNA-seq dataset. (b) Top GO terms of genes up- or down-regulated by introduction of NFIA into C2C12 myoblasts. The analyses were performed once based on the RNA-seq dataset. (c) Control and NFIA-expressing 3T3-F442A adipocytes were stained with Oil Red O seven days after inducing adipocyte differentiation. Scale bar, 50 μm. (d–f) Nfia (d), general adipocyte genes (e) and the brown-fat-specific genes (f) were quantified by RT-qPCR at the indicated time course (mean ± S.E.M.; N = 3 independent samples; ∗ p < 0.05, ∗∗ p < 0.01). (g) Immortalized, differentiated brown adipocytes were electroporated with a control siRNA or a siRNA for NFIA and stained with Oil Red O. Scale bar, 50 μm. (h–j) Nfia (h), Pparg (i), Cidea and Ucp1 (j) were quantified by RT-qPCR at the indicated time course (mean ± S.E.M.; N = 3 independent samples; ∗ p < 0.05, ∗∗ p < 0.01).
