FIG 4.
NO produced during hyperosmotic stress is proapoptotic. (A) Cell lysates were assayed for nitric oxide from wild-type MEFs treated with hyperosmotic medium and PKRi or control and shPKR MEFs for the indicated durations. (B) Cell lysates were assayed for arginine uptake from wild-type MEFs treated with hyperosmotic medium. (C) Caspase-3 cleavage was determined by quantification of immunoblot signals of cell extracts from control and shPKR MEFs treated with hyperosmotic medium and the nitric oxide scavenger cPTIO (100 μM) for the indicated durations. Proteins were detected at the following masses: PKR at 55 kDa, cleaved caspase-3 at 15 kDa, and tubulin at 55 kDa. (D) Cell survival was quantified using an MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay of MEFs treated with hyperosmotic medium and PKRi for the indicated durations. (E) Caspase-3 activities were measured in cell lysates from control and PKRi-treated MEFs as well as from control and shPKR MEFs treated with hyperosmotic medium for the indicated durations. (F) Protein synthesis levels were measured by [35S]Met-Cys incorporation in MEFs treated with PKRi and hyperosmotic medium for the indicated durations. Error bars represent standard errors of the means. *, P < 0.05; ns, not significant (Student's t test).