Extended Data Fig. 4. Celastrol does not affect home-cage, anxiety, or social behavior in DIO mice.

DIO mice were treated with vehicle or celastrol (100 μg/kg, i.p., once a day) for 3 days. On the fourth day, home-cage behavior (grooming, d,e), open field test (f-l) and social behavior (m-o) were assessed at ad libitum-fed condition. (a) Average 24-hour food intake per mouse during the treatment (n=3 cages for each group, P=0.0001). (b) Body weight (Day 0 vs. Day 4, P>0.99 for Vehicle-treated group and P=0.002 for celastrol-treated group) and (c) percent body weight reduction before (Day 0) and after 4 days (Day 4) vehicle- or celastrol-treatment (P<0.0001). (d) Frequency (numbers) of grooming (P=0.572). (e) Total time the mice spent grooming during home-cage behavior test (P=0.255). n=10 for both vehicle- and celastrol-treated groups. (f) Total distance traveled by the mice (P=0.956). (g) Average velocity of movement during the open field test (P=0.956). (h) Duration (% of time in the assay) the mice spent moving (P=0.999). (i) Frequency (numbers during assay) that mice traveled to the central portion of field (P=0.331). (j) Total time spent in the central portion of field (P=0.591). (k) Latency (in seconds) until initial entry into the central portion of open field (P=0.913). (l) Representative movement-tracking plots of vehicle- and celastrol-treated mice in the open field assay. n=8 for each group. (m) Total time the mice spent in the object-paired side chamber (object) versus novel mouse 1-paired side chamber (novel mouse 1). Object chamber vs. Novel mouse chamber, P=0.007 in vehicle-treated group and P<0.0001 in celastrol-treated group. (n) Total time the mice spent with object, which was in the left chamber and with novel mouse 1, and in the right chamber during the sociability test. Object vs. Novel mouse, P=0.0003 in vehicle-treated group and P<0.0001 in celastrol-treated group. (o) Total time the mice spent in the novel mouse 1-paired (novel mouse 1) or novel mouse 2-paired side chamber (novel mouse 2) during social novelty test (Novel mouse 1 vs. Novel mouse 2, P=0.01 in vehicle-treated group and P=0.02 in celastrol-treated group). n=8 for both groups during sociability test and n=7 for both groups during social novelty test. Values indicate average ± s.e.m. P values were determined by two-tailed Student t test (a, d-k) or two-way ANOVA with Bonferroni's multiple comparisons test (b,c, m-o). * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant (P>0.05).