Figure 4. Identification of ATF4 as a target of WTAP. AC16 cells were transduced with WTAP shRNA or WTAP-overexpressing plasmids and underwent H/R for 48 h. (A) MeRIP-qPCR analysis of ATF4 5′UTR m6A levels. (B–D) Expression of ATF4. (E) Analysis of TFEB 5′UTR showed a match to 5′-RRACU-3′ m6A consensus sequence. (F) Luciferase activity assay. (G) Binding of EIF3A to ATF4 mRNA was measured by RIP and qRT-PCR. (H, I) The ATF4 expression in AC16 cells transduced with EIF3A shRNAs. (J, K) Western blot analysis of ATF4 protein level upon CHX treatment in AC16 cells transduced with EIF3A shRNA. All experiments were repeated at least three times, and data are represented as mean ± SD. ***P < 0.001 vs control (untreated)/shNC. ##P < 0.01, ###P < 0.001 vs H/R.