Inhibition of PKR protects against mutations induced by age, IR, or a potent oncogene. The mutation frequency was measured serially in PB reticulocytes using the in vivo PIG-A mutation assay that detects the loss of the glycosylphosphatidylinositol-linked proteins like CD24 on the surface of reticulocytes by flow cytometry. The PIG-A mutation frequency was calculated as the percentage of CD24⁻ reticulocytes/total reticulocytes analyzed. (A) PB from unperturbed young (3-month-old) vs old (12-month-old) WT, PKRKO, and TgPKR mice was obtained, and reticulocytes isolated and analyzed by flow cytometry for the presence or absence of CD24. Increased PKR expression in TgPKR hematopoietic cells promotes an age-associated increase in the frequency of PIG-A mutation compared with WT or PKRKO. (B) NHD13-TgPKR mice, aged 9 months, have a significantly increased frequency of PIG-A mutation compared with NHD13 or NHD13-PKRKO mice. (C) Increased PKR expression promotes a significant increase in the frequency of PIG-A mutations following 5 Gy IR exposure. (D) PKRI treatment reduces the frequency of PIG-A somatic mutation in PB cells of mice following IR. WT mice were injected intraperitoneally with either 200 μg/kg PKRI or PBS (5 mice in each treatment group) and administered a single sublethal dose of IR (5 Gy). After IR, mice received either 200 μg/kg PKRI or PBS every 12 hours for 4 weeks. (E-F) NHD13 mice that were 4 months old were implanted with Alzet osmotic pumps (#1004) filled with either PKRI (1.5 mg/mL in PBS:dimethylsulfoxide) or vehicle control (PBS:dimethylsulfoxide). After 28 days, mice were euthanized to collect blood and BM. (E) The PIG-A mutation frequency in PB of NHD13 mice was significantly reduced by 28-day continuous PKRI treatment. (F) BM of NHD13 mice that received continuous PKRI treatment had significantly greater CFU-GEMM and total CFU activity. *P < .05; **P < .01.