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Fig. 1 ROS inhibit translation and activate the RSR. (A) U2OS cells were treated with increasing concentrations of menadione (25, 50, 150, 250, and 500 μM for 1 hour). Lysates were analyzed by immunoblotting with the indicated antibodies. (B) WT U2OS cells or U2OS cells deleted for ZAK (ΔZAK) were treated with a ZAK inhibitor (ZAKi, 2 μM) and menadione (Mena, 250 μM for 1 hour). Lysates were analyzed as in (A). (C) Schematic of ZAK protein isoforms. LZ, leucine zipper; SAM, sterile alpha-motif; S, sensor domain; CTD, C-terminal domain; SFBD, stress fiber binding domain. (D) U2OS cells were transfected with control (mock) small interfering RNA (siRNA) or siRNAs targeting the α or β isoforms of ZAK. Cells were treated with menadione (250 μM for 1 hour), and lysates were analyzed as in (A). (E) U2OS cells were pretreated with NAC (10 mM for 1 hour) followed by addition of menadione (250 μM for 1 hour) or anisomycin (Ani, 1 μg/ml for 1 hour) as indicated. Puromycin (10 μg/ml) was added to the culture 10 min before harvest, and lysates were analyzed by immunoblotting with the indicated antibodies. (F) U2OS cells, ΔZAK cells, and ΔZAK cells rescued with WT and mutated forms of ZAKα and WT ZAKβ were treated as in (D) [αΔΔ is a deletion of the “S” and “CTD” domain in (C)]. Lysates were analyzed as in (A). (G) U2OS cells were treated with menadione (250 μM for 1 hour) followed by washout (WO) in the presence of NAC (10 mM) for the indicated times. Cells were pulse treated with puromycin (10 μg/ml, 10 min) before harvest as in (E). Lysates were analyzed as in (A). (H) Cells from (B) were treated with ROS-generating β-lapachone (β-lapa, 20 μM for 1 hour) and lysates were analyzed as in (A). (I) U2OS cells were pretreated with NAC (10 mM for 1 hour), followed by addition of auranofin (Aura, 5 μM for 1 hour) as indicated. Lysates were analyzed as in (A). (J) Cells from (B) were treated with auranofin (5 μM for 1 hour), and lysates were analyzed as in (A).