Fig 2: BMS309403 reduced the release of profibrotic cytokines by inhibiting TGF-ß/Smad3 pathway and restored impaired FAO pathway in fibrotic kidney.a TGF-ß, p-Smad2, and p-Smad3 expression was verified in kidney samples by Western blot. Blots were stripped and reprobed for GAPDH. b RT-qPCR for TGF-ß, CTGF, PDGFB, and FGF2 transcripts normalized to Gapdh in kidney tissue. c The changes in PGC1a and PPAR? expression levels were confirmed in mouse kidneys by immunoblotting studies. d Ppargc1a, Pparg, Cpt1a, Cpt2, Acox1, and Acox2 mRNA expression were analyzed by RT-qPCR in total RNA extracts of kidneys from saline- or BMS309403-treated normal and UUO mice. The transcript normalized to Gapdh. Representative images from three independent experiments are shown above. *P < 0.05, **P < 0.01, ***P < 0.001, ns means no statistical significance.

Fig 3: miR-130b-3p specifically targets PPARG 3'UTR. (A) Detection of the abundance of miR-130b-3p by RT-qPCR after mimic-130b transfection in control cells. (B) mRNA expression analysis of PPARG after overexpressing miR-130b in control cells or knocking out miR-130b. (C) The binding site of miR-130b-3p seed sequence in 3'UTR region of PPARG and site-directed mutation of the binding site in PPARG 3'UTR. WT-PPARG represents the wild-type (WT) PPARG 3'UTR (42–48) sequence; MUT-PPARG represents the mutational PPARG 3'UTR sequence. (D) Detection of the luciferase activity in GMEC co-transfected with the miRNA (mimic-130b or mimic-NV) and dual-luciferase reporter plasmid (psi-WT-PPARG or psi-MUT-PPARG). (E,F) Detection of PPAR? protein abundance by Western blot after mimic-130b transfection in control cells. (G,H) Protein abundance detection of PPAR? in miR-130b KO and control cells. Data are displayed as mean ± SEM. p < 0.05, p < 0.01 and p < 0.001 are indicated as *, ** and ***, respectively.

Fig 4: Silencing the expression of PPARG inhibits the PGC1a expression in knockout cells. (A) Abundance detection of PGC1a mRNA by RT-qPCR after reducing PPARG expression. (B,C) Detection of the protein abundance of PGC1a in GMEC transfected with PPARG siRNA or NC. Data are displayed as mean ± SEM. p < 0.01 and p < 0.001 are indicated as ** and ***.

Fig 5: METTL3 is essential for m6A modification of Prdm16, Pparg, and Ucp1 transcripts.a–c The read density from m6A-RIP-seq experiments on BKO–flox/flox pairs showing the m6A peaks identified in the Prdm16, Pparg, and Ucp1 transcripts. d–f The m6A modification in Prdm16, Pparg, and Ucp1 transcripts in iBAT of 8-week-old Mettl3flox/flox and BKO mice were measured by m6ARIP-RT-qPCR (n = 3 for each group). g–j Primary brown preadipocytes seeded in 24-well plates were co-transfected with pMIR-REPORT Luciferase vectors (Prdm16, Pparg, Ucp1), siRNAs (Scramble siRNA, siMettl3-1, siMettl3-2) and ß-galactosidase (ß-Gal) reporter plasmid by X-tremeGENE siRNA Transfection Reagent for 24 h. Cells were then induced to differentiate for 48 h. METTL3 and GAPDH protein levels were measured by immunoblotting g. Relative Prdm16, Pparg, and Ucp1 luciferase activity were measured and normalized to ß-Gal levels h–j (Scr in h, n = 4; others, n = 5 biologically independent cell samples for each group). Data represent the mean ± SEM. Significance was determined by unpaired two-tailed Student’s t test analysis. *p < 0.05. **p < 0.01. Source data are provided as a Source Data file.