TBI selectively increases p16^Ink4a expression and SA-β-gal activity in LKS⁺ cells. (A) Quantification of p16^Ink4a mRNA expression in LKS^- and LKS⁺ cells from control (CTL) or irradiated (day 14 after TBI) mice. Top panel: Amplification profiles of representative real-time RT-PCR assays. Bottom panel: A minimal estimate of the fold increase in p16^Ink4a mRNA expression is presented as the mean ± SE of 3 amplification assays. (B) Quantification of p16^Ink4a-expressing cells in LKS^- and LKS⁺ populations by flow cytometry. The expression of p16 ^Ink4a in LKS^- and LKS⁺ cells from control (CTL) or irradiated (day 14 after TBI) mice was determined by flow cytometry as described in “Materials and methods.” Top panel: Representative flow cytometric analysis of the p16^Ink4a-expressing cells in LKS^- and LKS⁺ cell gates are shown. The numbers marked in the plots are the percentage of p16 ^Ink4a-positive cells. Bottom panel: The percentage of p16 Ink4a-positive cells is presented as the mean ± SE (n = 3). (C) Representative flow cytometric analysis of the p16^Ink4a-expressing cells in LKS⁺ subpopulation from irradiated (day 14 after TBI) and control Ink4a/Arf KO or wild-type mice. (D) Representative immunofluorescent (IF) staining of p16^Ink4a in sorted LKS^- and LKS⁺ cells from irradiated and control mice. (E) Representative SA-β-gal staining in sorted LKS^- and LKS⁺ cells from irradiated and control mice. ^*P < .05 and ^***P < .001, compared with control unirradiated cells.