Extended Data Fig. 6. Celastrol fails to increase STAT3 phosphorylation and gene expression in the hypothalamus of Il1r1^−/− mice.

Il1r1^+/+ and Il1r1^−/− male mice were fed a HFD for 20 weeks and then administered either vehicle (Veh) or celastrol (Cel, 100 μg/kg, i.p.) daily for 3 days. Each group of mice subsequently received a single dose of vehicle or celastrol (200 μg/kg, i.p.) on the morning of the fourth day and was then fasted for 6 hours prior to extraction of the hypothalamus. Phosphorylation of STAT3 (p-STAT3^Tyr705) in the medial basal hypothalamus (MBH) was analyzed by immunofluorescence staining using a phospho-specific antibody. (a, c, e) Representative images of p-STAT3^Tyr705 immunostaining in the (a) arcuate nucleus (ARC), (c) ventromedial hypothalamus (VMH) and (e) dorsomedial hypothalamus (DMH) of Il1r1^+/+ and Il1r1^−/− mice after 4 days of vehicle or celastrol treatment. (b, d, f) Quantitation of total p-STAT3^Tyr705-positive cell number and total fluorescence intensity of p-STAT3^Tyr705 in the (b) ARC (Il1r1^+/+-Veh vs. Il1r1^+/+-Cel, P=0.0007, and Il1r1^+/+-Cel vs. Il1r1^−/−-Cel, P=0.006 for cell number. Il1r1^+/+-Veh vs. Il1r1^+/+-Cel, P=0.001, and Il1r1^+/+-Cel vs. Il1r1^−/−-Cel, P=0.008 for fluorescence intensity). (d) VMH (Il1r1^+/+-Veh vs. Il1r1^+/+-Cel, P=0.0003, and Il1r1^+/+-Cel vs. Il1r1^−/−-Cel, P=0.0006 for cell number. Il1r1^+/+-Veh vs. Il1r1^+/+-Cel, P=0.0004, and Il1r1^+/+-Cel vs. Il1r1^−/−-Cel, P=0.0007 for fluorescence intensity). (f) DMH (Il1r1^+/+-Veh vs. Il1r1^+/+-Cel, P=0.002, and Il1r1^+/+-Cel vs. Il1r1^−/−-Cel, P=0.02 for cell number. Il1r1^+/+-Veh vs. Il1r1^+/+-Cel, P=0.004, and Il1r1^+/+-Cel vs. Il1r1^−/−-Cel, P=0.01 for fluorescence intensity). The experiments a-f were repeated in two independent cohorts with similar outcomes and the results in b, d and f represent the combination of two independent experiments (total n=7 for both vehicle- and celastrol-treated mice in Il1r1^+/+ group; n=8 for both vehicle- and celastrol-treated mice in Il1r1^−/− group). Scale bars, 100 μm. 3V, third ventricle. (g-j) Expression levels of genes in the hypothalamus of Il1r1^+/+ and Il1r1^−/− mice treated with vehicle or celastrol (100 μg/kg, i.p., once a day) for 4 days. (g) Agrp (Il1r1^+/+-Veh vs. Il1r1^+/+-Cel, P=0.005 and Il1r1^−/−-Veh vs. Il1r1^−/−-Cel, P>0.99), (h) Npy (Il1r1^+/+-Veh vs. Il1r1^+/+-Cel, P=0.362 and Il1r1^−/−-Veh vs. Il1r1^−/−-Cel, P=0.871), (i) Pomc (Il1r1^+/+-Veh vs. Il1r1^+/+-Cel, P=0.757 and Il1r1^−/−-Veh vs. Il1r1^−/−-Cel, P>0.99) and (j) Socs3 mRNA (Il1r1^+/+-Veh vs. Il1r1^+/+-Cel, P=0.03 and Il1r1^−/−-Veh vs. Il1r1^−/−-Cel, P>0.99). (n=6 for vehicle- and n=7 celastrol-treated mice in Il1r1^+/+ group; n=4 for both vehicle- and celastrol-treated mice in Il1r1^−/− group). Values indicate average ± s.e.m. P values were determined by two-way ANOVA with Bonferroni's multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant (P>0.05).