Fig. 2
From: SYK kinase mediates brown fat differentiation and activation
SYK mediates expression of characteristic brown fat genes and activates the AKT/mTOR pathway. a Representative immunoblots of SYK protein expression from three independent differentiations of in vitro differentiated brown adipocytes from CreERT2 Sykflox/flox mice on day 8 of differentiation following continuous treatment with 4-hydroxy tamoxifen (4-OHT) for 9 days. b RNA-seq was conducted on 3 replicates of 4-OHT treated Sykflox/flox (WT) and CreERT2 Sykflox/flox (KO) cells as shown in a before and after isoproterenol treatment at day 8 of differentiation. Log fold change in WT cells before and after induction is shown on the x axis and log fold change in KO cells before and after induction is shown on the y axis. Genes shown had at least a 1.5-fold up- or down-regulation in WT or KO cells after stimulation with an FDR < 0.05. Genes with a fold change difference of 1.5 higher in WT compared to KO with a p value < 0.05 are shown in red. c Enriched GO categories with a Benjamini adjusted p value < 0.05 for genes shown in red in b. d Fold change of mRNA expression in day 8 differentiated primary brown adipocytes with tamoxifen-induced reduction in SYK protein as described in (a) and stimulated with isoproterenol. e Representative immunoblots of two independent experiments of primary brown adipocytes stimulated with isoproterenol on day 8 of differentiation for 48 h in complete medium in the presence or absence of 5 μM SYK-i3. f Day 8 differentiated brown adipocytes (prim, primary; imm, immortalized) stimulated with isoproterenol and pretreated with mTORC inhibitors torin 1 (10 μM), torin 2 (10 μM) or KU0063794 (10 μM), AKT inhibitor API-2 (10 μM), or PKA inhibitors PKI 14-22 (10 μM), or H89 (10 μM) (n = 3). g Representative immunoblots of three independent experiments of day 8 primary brown adipocytes pretreated with 5 μM SYK-i3 and stimulated with isoproterenol for 5 min. pPKAsub, antibody recognizing PKA substrate phosphorylation motif RRXS*/T*. For f, mean % ± SEM and d, fold change vs unstimulated control ± SEM was calculated. For d, two-tailed unpaired Student’s t test was performed (*P < 0.05, **P < 0.005 and ***P < 0.0005) and for f, a one-way ANOVA was performed
