Extended Data Fig. 6: Celastrol fails to increase STAT3 phosphorylation and gene expression in the hypothalamus of Il1r1–/– mice.
From: IL1R1 is required for celastrol’s leptin-sensitization and antiobesity effects
Il1r1+/+ and Il1r1–/– male mice were fed a HFD for 20 weeks and then administered either vehicle (Veh) or celastrol (Cel; 100 μg/kg body weight, i.p.) daily for 3 d. Each group of mice subsequently received a single dose of vehicle or celastrol (200 μg/kg body weight, i.p.) on the morning of the fourth day and was then fasted for 6 h prior to extraction of the hypothalamus. Phosphorylation of STAT3 (p-STAT3Tyr705) in the medial basal hypothalamus (MBH) was analyzed by immunofluorescence staining using a phospho-specific antibody. a,c,e, Representative images of p-STAT3Tyr705 immunostaining in the arcuate nucleus (ARC) (a), ventromedial hypothalamus (VMH) (c), and dorsomedial hypothalamus (DMH) (e) of Il1r1+/+ and Il1r1–/– mice after 4 d of vehicle or celastrol treatment. b,d,f, Quantification of total p-STAT3Tyr705-positive cell numbers and total fluorescence intensity for p-STAT3Tyr705 in the 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) (b), 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) (d), and 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) (f). The experiments in 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 mice for both vehicle- and celastrol-treated mice in the Il1r1+/+ group; n = 8 for both vehicle- and celastrol-treated mice in the 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 body weight, i.p., once a day) for 4 d, including for Agrp (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.005; Il1r1–/––Veh vs. Il1r1–/––Cel, P > 0.99) (g), Npy (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.362; Il1r1–/––Veh vs. Il1r1–/––Cel, P = 0.871) (h), Pomc (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.757; Il1r1–/––Veh vs. Il1r1–/––Cel, P > 0.99) (i), and Socs3 (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.03; Il1r1–/––Veh vs. Il1r1–/––Cel, P > 0.99) (j) (n = 6 vehicle-treated and n = 7 celastrol-treated mice in the Il1r1+/+ group; n = 4 vehicle- and celastrol-treated mice in the Il1r1–/– group). Values indicate averages ± 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).
