FIG 3.

PKR is necessary for maximal induction of iNOS mRNA. RT-qPCR analysis was performed of RNA isolated from control and shPKR MEFs treated with 600 mosM sucrose (A, F, and G) or from MEFs treated with PKRi for 1 h prior to addition of hyperosmotic medium (B) for the indicated durations. (C) Schematic representation of wild-type and κB-site mutant plasmid constructs of the iNOS promoter fragments fused to the luciferase reporter gene. (D) Luciferase activities were normalized to Renilla luciferase in MEFs transfected with the constructs shown in panel C and then treated with hyperosmotic medium for the indicated durations. (E) Control and shPKR MEFs were transfected with the wild-type luciferase reporter and treated with hyperosmotic medium for the indicated durations. Luciferase activity was normalized to that of the transfected Renilla luciferase reporter. (H) A model of iNOS mRNA amplification under hyperosmotic stress via PKR activation and NF-κB signaling. In all panels mRNA levels were normalized to the level of GAPDH. Error bars represent standard errors of the means. *, P < 0.05; ns, not significant (Student's t test).