Puma is up-regulated by γ-irradiation in a gene dose-dependent manner and is selectively expressed in LSK cells. (A) QPCR analysis of Puma mRNA expression levels in primary hematopoietic cells from Puma^+/+ and Puma^+/− mice at 1.5 and 3 hours after irradiation (5 Gy) or no treatment. Puma gene expression levels were normalized, based on the levels of the housekeeping gene HPRT. The cross comparison between Puma^+/+ and Puma^+/− mice at each time point was highly significant (2-tailed Student t test). *P < .05, ***P < .001 (Puma^+/+ vs Puma^+/− at each time point). (B) Western blot analysis of Puma expression levels. Lysates extracted from BM of Puma^+/+ and Puma^+/− mice at the indicated time points after irradiation (5 Gy) were probed with an anti-Puma and an anti–β-actin antibody (loading control). (C) QPCR analysis of PUMA induction by irradiation in LSK cells. LSK and Lin⁺ cells were sorted, respectively, from wild-type mice and cultured in the StemSpan serum-free expansion medium (10 ng/mL interleukin-3). The cells were untreated or treated with γ-irradiation (7 Gy) and then subjected to total RNA extraction at 3 hours after irradiation. QPCR analysis was used to qualify Puma mRNA level in nonirradiated and irradiated cells and normalized to HPRT expression level. ***P < .001 and *P < .05 (LSK vs Lin⁺ cells). Data in panels A and C are mean ± SD.