Fig. 5.

m⁶A reader protein YTHDF1 promotes autophagy activation and BECN1 mRNA stability via recognizing the m⁶A binding site. HSC-T6 and HSC-LX2 cells were added sorafenib (10 μM), erastin (10 μM), and RSL3 (2.5 μM) and treated for 24 h. (A) Western blot was used to assay the protein levels of m⁶A readers (n = 3 in every group). (B) The mRNA levels of m⁶A readers were determined by real-time PCR (***, p < 0.001, n = 3 in every group). (C) YTHDF1 plasmid or control vector were transfected into HSC-LX2 cells and treated with erastin (10 μM) for 24 h, and then were pretreated with Act-D (5 μg/ml) for indicated times. Real-time PCR was used to measure the remaining BECN1 mRNA levels (*, p < 0.05, n = 3 in every group). (D) YTHDF1 plasmid or control vector were transfected into HSC-LX2 and treated with erastin (10 μM) for 24 h. Western blot was used to determine the protein levels of BECN1 at different times under the CHX (100 μg/ml) treatment (n = 3 in every group). (E) The binding of YTHDF1 and BECN1 mRNA was measured by ribonucleoprotein immunoprecipitation (RNP IP) (**, p < 0.01, n = 3 in every group). (F) biotinylated transcripts of the BECN1 mRNA 3′-UTR, CDS, 5′-UTR were used for mRNA affinity isolation assay (n = 3 in every group). (G) HSC-LX2 cells were transfected with control vector or YTHDF1 plasmid and by erastin (10 μM) treatment for 24 h. The levels of m⁶A modification in BECN1 mRNA 3′-UTR, CDS and 5′-UTR were determined by MeRIP qPCR (**, p < 0.01, n = 3 in every group). (H) HSC-LX2 cells were stably transfected with empty vector (pGL3)or luciferase constructs carrying the genes encoding 5′-UTR-BECN1, CDS-BECN1, 3′-UTR-BECN1. Then cells were added erastin (10 μM) and treated for 24 h, and luciferase activities were measured (**, p < 0.01, n = 3 in every group). (I) BECN1 mRNA m⁶A binding site was showed. (J) Schematic representation of positions of m⁶A motifs and mutation in CDS within BECN1 mRNA were showed. (K) Binding of YTHDF1 with the BECN1-CDS-WT, BECN1-CDS-Mut1 (A437G) and BECN1-CDS-Mut2 (A1276G) in HSC-LX2 were analyzed by YTHDF1 RNP IP (**, p < 0.01, n = 3 in every group). (L) HSC-LX2 cells were transfected with control vector or YTHDF1 plasmid and BECN1-CDS-WT, BECN1-CDS-Mut1 and BECN1-CDS-Mut2 plasmid, and then were pretreated with Act-D (5 μg/ml) for indicated times. Real-time PCR was used to measure the remaining BECN1 mRNA (*, p < 0.05, n = 3 in every group). (M) After transfected with BECN1-CDS-WT, BECN1-CDS-Mut1 and BECN1-CDS-Mut2 plasmid, HSC-LX2 cells were treated with erastin (10 μM) for 48 h. The protein expression of BECN1 was measured by western blot (*, p < 0.05, ***, p < 0.001, n = 3 in every group).