Extended Data Fig. 2: EPAC1 signals through ERK1/2 and upregulates C/EBPβ in brown preadipocytes.

a, Relative glycerol release of mature BA acutely stimulated with 007, 6-MB or NE (norepinephrine/noradrenaline), n = 3 independent experiments. b, Relative CDK1 phosphorylation on Y15 upon 007, 6-MB stimulation, n = 4 biologically independent samples c, supervised hierarchical clustering of ERK1/2 substrates. Position of phosphorylation, multiplicity of phosphopeptide indicated, * marks regulatory sites (red =activating, blue=inhibiting). n = 4 biologically independent samples. d, Representative immunoblot for ERK1/2 phosphorylation (left panel) and quantification (right panel) of brown preadipocytes stimulated with 007 ± U0126, n = 4 independent experiments. e, Representative immunoblot for ERK1/2 phosphorylation (left panel) and quantification (right panel) of brown preadipocytes isolated from Rapgef3^-/- and WT mice n = 4 biologically independent samples. f, Dusp1 expression in brown preadipocytes upon 007, 6-MB stimulation, n = 3 independent experiments. g, Representative immunoblot for ERK1/2 phosphorylation (left panel) and quantification (right panel) of brown preadipocytes stimulated with 007 or 6-MB for 48 h, n = 3 independent experiments. h, Oil RedO Staining (left panel) and quantification of Ucp1 mRNA levels of BA differentiated with 007 ± U0126, n = 3 independent experiments. i, Quantification of proliferation of primary murine brown preadipocytes isolated from BAT of Rapgef3^-/- and WT mice, n = 4 biologically independent samples. j, Quantification of DAPI-positive BA after differentiation ± 007 n = 3 independent experiments. k, Quantification of primary murine proliferating cells pretreated ± Rapamycin and stimulated ± 007 or ± NE, n = 4 biologically independent samples. l-n, BA stimulated during the indicated times with 007. n = 4 independent experiments l, Quantification of lipid droplet accumulation of BA. For the box plots, the centre line shows the median, the box limits show the first and third quartiles, the upper and lower whiskers extend from the maximum to the minimum value. m-n, relative qPCR expression of the BA differentiation markers Pparg and Ucp1. o, Representative immunoblot (left) and quantification (right) of C/EBPβ in Rapgef3^-/- BA induced ± 007, n = 5 independent experiments. p, Representative immunoblot (left) and quantification (right) of BA transduced with a LV expressing different CRISPR/Cas9 constructs to knock down C/EBPβ, or a non-targeting control construct (LV-NTC), n = 3 biologically independent samples. q-r, Relative expression of differentiation markers in BA transduced with LV-NTC or Crispr construct #2 targeting C/EBPβ, and treated with ± 007 throughout differentiation, n = 3 biologically independent samples. s, C/EBPβ expression of brown preadipocytes treated with ± 007 ± rolipram, n = 3 independent experiments. t, C/EBPβ expression of brown preadipocytes treated with ± 007 ± rolipram ± U0126, n = 3 independent experiments. P values determined by one-way ANOVA (Tukey’s multiple comparison test), unpaired two-tailed t-test in e, i, j, and one-way RM ANOVA (Holm-Šídák’s multiple comparison test) in l-n. Data are presented as mean values ± SEM. Source numerical data and unprocessed blots are available in source data.

Source data