Fig. 4

PSAT1 was modulated by METTL3-mediated m6A methylation. (A) SRAMP predicted that PSAT1 mRNA has an m6A site. (B and C) METTL3 and PSAT1 mRNA expression were examined using in ARPE-19 cells transfected with sh-NC, sh-METTL3, Vector, or OE-PSAT1 using RT-qPCR assay. (D and E) Western blot analysis of METTL3 and PSAT1 protein level in ARPE-19 cells transfected with sh-NC, sh-METTL3, Vector, or OE-PSAT1. (F) Changes in the m6A methylation level of PSAT1 after METTL3 knockdown or overexpression were assessed using MeRIP-qPCR assay. (G) Their binding was verified using a luciferase reporter assay in 293t cells. (H) Influences of METTL3 downregulation or upregulation on PSAT1 mRNA stability after Actinomycin D treatment was assessed using RT-qPCR in ARPE-19 cells. (I) METTL3 content was measured in the serum of 28 DR and 28 Control group using RT-qPCR assay. (J) Pearson correlation analysis was applied to evaluate the expression association between METTL3 and PSAT1 in DR patients. (K and L) PSAT1 mRNA level and protein level were examined in ARPE-19 cells treated with NG, MA, and HG using RT-qPCR assay and Western blot. *P < 0.05, **P < 0.01, ***P < 0.001, n = 3