Fig 2: FTO alters CD36 expression. (A,B) Silencing efficiency of FTO. (A) qRT-PCR analysis of the effect of transfection of FTO siRNA after 24 h in AC16 cells. ***p < 0.001, compared with the NC groups, n = 3. (B) Western blotting analysis of the effect of transfection of FTO siRNA after 48 h in AC16 cells. **p < 0.01, compared with the NC group, n = 4. (C) Effect of FTO loss of function and LHD co-treatment on CD36 expression in AC16 cells. ***p < 0.001, n = 3. (D) Effect of FTO loss of function and LHD co-treatment on IL-6 and TNF-α expression in AC16 cells. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3. (E,F) Efficiency of FTO overexpression. (E) Expression of FTO mRNA after FTO overexpression for 24 h in AC16 cells. (F) Expression of FTO protein after FTO overexpression for 48 h in AC16 cells. ***p < 0.001, compared with the CTL group, n = 3. (G) Effect of FTO overexpression on the expression of CD36 in PA-treated AC16 cells. *p < 0.05, **p < 0.01, n = 3. (H) Effect of FTO overexpression on the expression of IL-6 and TNF-α in PA-treated AC16 cells. **p < 0.01, ***p < 0.001, n = 3.

Fig 3: Fto-knockout mice harbored a specific gut microbial signature to suppress inflammation. (A) PCA revealed a trend of separation by genotype (WT, n=8; KO, n=8). (B) Microbial distribution at the genus level. (Lactobacillus is marked in red). (C) Relationship of OTUs with different abundances. Lactobacillus (OTU3) was negatively correlated with almost all other OTUs with different relative abundances observed between the two groups. (D) Correlation of Lactobacillus abundance with behavioral indices. EPM: elevated plus maze; FST: forced swim test; KO: knockout; OFT: open-field test; OTU: operational taxonomic unit; PCA: principal component analysis; TST: tail suspension test; WT: wild-type.

Fig 4: LHD binds the FTO protein and regulates the level of m6A modifications. (A) The top 15 compounds most likely to bind LHD. (B) Docking models simulating LHD binding into the FTO crystal structure (PDB ID: 3LFM). (C) Representative western blotting analysis of FTO from the DARTS assay. AC16 cells lysates with 83.4 (LHD L), 166.7 (LHD M), and 250.0 (LHD H) µM LHD were incubated for 1 h at room temperature before pronase digestion, n = 3. (D) Western blotting analysis of the thermal stability of FTO in LHD-treated cells, n = 3. (E) Structure of the complex of FTO bound with LHD; the yellow dotted lines indicate hydrogen bonding. (F) Determination of the abundance of the modification in 25 µM LHD-treated cells after 16 h, n = 4.

Fig 5: Fto deficiency-induced hyposensitivity to anxiety and depression is largely dependent on gut microbiota. (A) Fto heterozygous (HZ) mice spent more time on the open arms during the EPM test after CUMS. Our analyses confirmed that these changes were not due to alterations in basal locomotor activity. However, the difference between the two groups was reduced following antibiotic treatment. There was no significant difference in open arm preference. (B) Fto HZ mice spent more time in the central area during the OFT. Our analyses again confirmed that these changes were not due to alterations in basal locomotor activity, and that the difference between the two groups was reduced following antibiotic treatment. There was no significant difference in fecal pellets. (C) Fto HZ mice spent less time being immobile during the FST, although the difference between the two groups was reduced following antibiotic treatment. (D) There was no difference in immobility time during the TST between the groups. Data are presented as mean±s.e.m. The multi-factor analysis of variance(ANOVA; genotype×treatment) revealed no significant interaction between factors for Time on open arms, Time in center and immobility time in FST, TST. Arm entries and Distance travelled are covariates for time on open arms and time in center respectively. It revealed a significant effect of genotype for time on open arms (P<0.0001), time in center (P=0.03), immobility time in FST (P=0.01) and a significant effect of treatment for time on open arms(P=0.001), open arms preference(P<0.0001), fecal pellets(P<0.0001), immobility time in FST(P=0.006). *P< 0.05 **P<0.01 ***P<0.001 Difference between the two groups was measured according to independent samples t-test. Abs: Antibiotics; CUMS: chronic unpredictable mild stress; EPM: elevated plus maze; FST: forced swim test; IT: Immobility Time; OFT: open-field test; TST: tail-suspension test.