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Fig. 2 ROS-induced ZAK? activation is associated with ribosome stalling and collision. (A) U2OS cells were treated with menadione (Mena, 250 ?M for 30 min) and ISRIB (200 nM). Lysates were digested with MNase and separated on a linear sucrose gradient. Arrows highlight UV peaks that are indicative of increased ribosome collision. (B) U2OS cells were pretreated with menadione (250 ?M for 1 hour) as indicated and harringtonine (HTN, 2 ?g/ml) for the indicated times to induce ribosome run-off. Puromycin (10 ?g/ml) was added to the culture for 5 min before harvest (but after HTN incubation time) and lysates were analyzed by immunoblotting with the indicated antibodies. (C) U2OS cells were treated with menadione (250 ?M for 1 hour) and inhibitors (i, 1 ?M) against PERK and GCN2 as indicated. Lysates were analyzed as in (B). (D) Schematic of tripartite in vitro translation (IVT) approach. Ribosomes, ribosome-free cytoplasm, and mRNA can be individually treated before combination. (E) The three fractions from (D) were individually treated with hydrogen peroxide (10 min) and neutralized by the addition of catalase as indicated. Translation efficiency in the combined reaction was determined by luciferase assay. (F) As in (E), except that the fractions were individually irradiated with UVB (500 J/m2) before in vitro translation. (G) Schematic of modified tripartite IVT assay. In contrast to (D), HeLa cells were pretreated with NAC (10 mM for 1 hour), followed by addition of menadione (250 ?M for 1 hour) before purification of fractions. (H) Combined IVT reactions with treated with ribosome-free cytoplasm from (G). (I) As in (H), except that treated ribosomes from (G) were used. All values indicate luciferase activity normalized to the control. For (E), (F), (H), and (I), data are plotted as means, and all error bars represent the SEM (n = 3 biological replicates). ns, nonsignificant; *P ? 0.05; **P ? 0.01; ***P ? 0.001; ****P ? 0.0001 by Student?s t test for two groups and one-way ANOVA with Tukey?s post hoc test for more than two groups. (J) Whole-cell RNA isolated from HeLa cell fractions from (G) was separated on urea agarose gel and stained for RNA. nt, nucleotides. (K) Northern blot for tRNA-Arg-TCT on RNA samples from (J). Schematic of tRNA intermediates corresponding to distinct bands are shown on the left side of the blot.