Fig. 6: mNAT10 promotes adipogenesis via mKLF9 mRNA ac4C modification.

A NAT10 RIP-PCR revealed that mNAT10 can interact with mKLF9 (n = 4). B NAT10 RIP-PCR revealed that hNAT10 can interact with hKLF9 (n = 4). Dual-luciferase reporter assay verified that mNAT10 is combined with 3 motifs of mKLF9 in 3T3-L1 cells (n = 3) (C); hNAT10 combined with 1 motif of hNAT10 in HEK293T cells (n = 3) (D); hNAT10 ac4C modification site (G641E) mutation did not combine with hKLF9 in HEK293T cells (n = 3) (E). F–K hNAT10 ac4C modification site (G641E) mutation did not affect hKLF9, hPPARG, hCEBPA and hCEBPB (n = 3). L RNA decay experiment detected the mKLF9 mRNA stability after 3T3-L1 cells were silenced by mNAT10 for 48 h (n = 4). M, N The rescue experiments revealed that silencing of mKLF9 is able to partially decrease cellular lipid droplets form (Oil red O staining) (n = 3), and cellular TG content (n = 3). O, P The qPCR experiment revealed that the silencing of mKLF9 resulted in a reduction in the mRNA expression levels of both mCEBPA and mPPARG, subsequent to the overexpression of mNAT10 in 3T3-L1 cells (n = 3). Q–V The western blot experiment revealed that the silencing of mKLF9 resulted in a reduction in the protein expression levels of both mCEBPA, mCEBPB and mPPARG, subsequent to the overexpression of mNAT10 in 3T3-L1 cells (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.