Figure 3.

Role of ALKBH5 in ATF4 mRNA methylation level. (A) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis. Hep3B were treated with sorafenib (SOR; 10 μM) for two hours or left untreated (unt), lysed, and their extracts were subjected to MeRIP using m⁶A antibodies. Immunoprecipitated m⁶A RNAs are then quantified by RT-qPCR using oligos specific to the main ORF of ATF4 mRNA. The amounts of m⁶A ATF4 mRNA were normalized against IgG precipitate and then expressed relative to untreated conditions. ** p ≤ 0.01. (B) Schematic representation of the 5’UTR ATF4 RNA reporter. (C) MeRIP-qPCR analysis of m⁶A level of the biotinylated 5′UTR ATF4 RNA reporter incubated with protein extracts prepared from either untreated (unt) or sorafenib (SOR)-treated Hep3B. RNA is then subjected to MeRIP using m⁶A antibodies. Immunoprecipitated m⁶A RNA is then incubated with streptavidin-agarose beads to purify the biotinylated reporter RNA, which is quantified by RT-qPCR using oligos specific to the 5′end of ATF4 mRNA. The amounts of m⁶A ATF4 reporter RNA were normalized against IgG precipitate and then expressed relative to the mock condition. Data are representative of 3 separate experiments, and values are given as mean ± SD. ** p ≤ 0.01, *** p ≤ 0.001, ns: not significant. (D) MeRIP-qPCR analysis of m⁶A level of 5′UTR ATF4 RNA reporter incubated with proteins extracts from Hep3B stably expressing either a control shRNA (shNT) or shALKBH5 and treated with SOR (10 µM, 2 h). Depletion of ALKBH5 is validated by western blot using specific antibodies as described in Figure 1 (left panels). m⁶A methylated ATF4 reporter RNAs were isolated and quantified by RT-qPCR (right graphs) as above. The amounts of m⁶A ATF4 reporter RNA were normalized against IgG precipitate and then expressed relative to the shNT condition. Data are representative of 3 separate experiments, and values are given as mean ± SD. ** p ≤ 0.01, ns: not significant. Original images can be found in Supplementary Materials.