Fig. 5
SMBJ improves Leydig cell function by upregulating METTL3 and affecting the PI3K/AKT pathway. (A) and (B) METTL3 was successfully knockdown using small interfering RNA. The data from the Western blot analysis of METTL3 are presented as the means ± standard deviations (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) The effect of different concentrations of high glucose on R2C cell, and (D) The effect of different concentrations of SMBJ on R2C cell (L:10 µl/ml, M:50 µl/ml, H:100 µl/ml). (E), (F), (G) and (H) METTL3 knockdown attenuated the upregulation of METTL3 caused by SMBJ at the protein level. Western blot results for METTL3, P-AKT/AKT, and P-PI3K/PI3K. The data shown are the means ± standard deviations (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (I) Total m6A levels following METTL3 knockdown in R2C cell (n = 3). (J) MeRIP-qPCR showing reduced m6A modification in PI3K mRNA upon METTL3 knockdown (n = 3). (K) MeRIP-qPCR showing reduced m6A modification in AKT mRNA upon METTL3 knockdown (n = 3). (L) and (M) Stability of PI3K/AKT mRNA after the inhibition of transcription by actinomycin D.
