Fig. 1.
Identification of kinases regulating Ucp1 expression in response to β-adrenergic stimulation. a Screening of a kinase inhibitor library (n = 90) to identify small molecules modulating Ucp1 RNA expression in response to stimulation with isoproterenol in in vitro differentiated day 8 immortalized brown adipocytes. Blue, pan-kinase inhibitors; red, SYK inhibitor ER27319; dotted line denotes + isoproterenol control (n = 3). b Representative immunoblots of two independent in vitro differentiations of primary brown adipocytes (b) and of two independent BAT isolations with two mice each (c) of SYK and pathway members as well as brown adipocyte markers. d Immunofluorescence of primary brown adipocytes stimulated with isoproterenol in the absence or presence of 2 µM SYK-i3 (R406), one representative IF of five independent replicates is shown. Scale bar = 10 µm. e Ucp1 RNA expression after stimulation with isoproterenol of differentiated day 8 immortalized brown adipocytes pretreated with 10 μM SYK inhibitors (SYK-i1 is ER27319; SYK-i2 is Bay 61-3606) or following transfection with siRNA targeting Syk mRNA 4 days prior (si1-Syk and si2-Syk) (n = 3). f Isoproterenol-induced Ucp1 RNA expression in primary day 8 brown adipocytes in vitro pretreated with SYK-i3 (R406) or in primary day 8 brown adipocytes derived from CreERT2 Sykflox/flox mice (KO) or Sykflox/flox (WT) both treated with 4-hydroxy tamoxifen (4-OHT) one day prior to induction of differentiation (n = 3). g Ucp1 RNA expression in primary day 8 brown adipocytes after overexpression of wild-type mouse SYK (Syk-OE1 and Syk-OE2) or pretreatment with 1 μM phosphatase inhibitor 3AC and stimulation with isoproterenol (n = 2). h Oxygen consumption rate (OCR) of immortalized brown adipocytes stimulated with isoproterenol and pretreated with 10 μM (SYK-i2) or following siRNA mediated knock down of Syk 4 days prior to measurement (si1-Syk, si2-Syk). Results are shown as average OCR + SEM of 18 measurements per time point and representative of three replicates. i Cell culture medium glycerol content from immortalized brown adipocytes at day 8 treated with isoproterenol in the absence or presence of 10 µM SYK-i2 (n = 3). For a, e–g, mean % of iso stimulated control ± SEM. For e, g, i, two-tailed unpaired Student’s t test was performed (*P < 0.05, **P < 0.005 and ***P < 0.0005). For f, two-tailed unpaired Student’s t test was performed on Log transformed values