Figure 1.

The RNA demethylases ALKBH5 and FTO are required for SOR-induced ATF4 expression. (A) Conserved consensus m⁶A site in the 5′UTR of ATF4 mRNA. R = A or G; H = A, C, or U, as described [18,19,20]. * corresponds to the conserved nucleotides, including the methylated adenosine in bold. (B–D) Hep3B was treated with 10 µM SOR for two hours. (B) Left panels: Cells were harvested and lysed, and protein extracts were analyzed by western blot for the expression of ALKBH5, ATF4, p-eIF2α, pan-eIF2α, and tubulin (Tub; loading control) using the corresponding antibodies. Right panel: The expression level of ATF4 was estimated by densitometry quantification of the film signal using Image Studio™ Lite Software (version 4.0.21) and standardized against total tubulin. **** p ≤ 0.0001 (Student’s t-test). (C) Left panels: Cells were harvested and lysed, and protein extracts were analyzed by western blot for the expression of the indicated proteins using the corresponding antibodies. Tubulin serves as a loading control. Right panel: The expression level of ATF4 was estimated by densitometry quantification of the film signal using Image Studio™ Lite Software and standardized against total tubulin. *** p ≤ 0.001; **** p ≤ 0.0001 (Student’s t-test). (D) Total RNA was isolated from harvested Hep3B, and the level of ATF4 mRNA relative to GAPDH mRNA was quantified by real-time q(RT)-PCR using the ΔΔCt method. The presented results are the mean of at least triplicate measurements, with error bars corresponding to the S.D. (E,F) Hep3B were treated with 10 µM SOR for twenty-four hours. (E) Clonogenic survival assays. After treatment, cells were trypsinized, counted, and seeded in the absence of the drug and incubated for 10 days. Populations > 20 cells were counted as one surviving colony. Data were calculated as the percentage of surviving colonies relative to the number found in plates corresponding to mock-depleted cell plates. Data shown are representative of at least 3 separate experiments, and values are given as mean ± SD. (Student’s t-test). ** p ≤ 0.01; * p ≤ 0.05. (F) Cell viability, assessed by MTT assay, shows the viability of Hep3B cells stably expressing shALKBH5 or shFTO after exposure to SOR for twenty-four hours. Data shown are representative of at least 3 separate experiments, and values are given as mean ± SD. (Student’s t-test). ** p ≤ 0.01; **** p ≤ 0.0001. Original images can be found in Supplementary Materials.