Fig. 2
B-1p cells from middle-aged mice survive and proliferate longer in cell culture after 72h and present radioresistance capacity after 3.5Gy of gamma-ionization. A Absolute number of live B-1p cells (PI-). Two-way ANOVA F(3,8)=13.2 p=0.002. Differences are observed between young 24h (1.030 ± 0.29) and young 72h (0.173 ± 0.064; p<0.001), young 24h and aged 24h (0.570 ± 0.118; p<0.001), young 24h and aged 72h (0.491 ± 0.1043; p=0.002), young 72h and aged 72h (p=0.036) and young 72h with aged 24h (p=0.020). Red line represents the initial number of seeded cells (3x103). B Absolute number of live cells in proliferation (levels of median celltrace violet fluoresence intensity). Two-way ANOVA F(3,8)=18.7 p<0.001. Differences are observed between young 24h (1.028 ± 0.99) and young 72h (0.130 ± 0.05; p<0.001), young 24h and aged 24h (0.380 ± 0.07; p<0.001), young 24h and aged 72h (0.323 ± 0.005; p<0.001), young 72h and aged 72h (p=0.038) and young 72h with aged 24h (p=0.011). Red line represents the initial number of seeded cells (3x103). C Absolute number of live cells (Annexin-7AAD-) after 72h in co-culture with OP9 cells. This represents the relation between cell number counted after culture and the percentage of cells annexin-7AAD- (Additional file 1: Fig. S2). One-way ANOVA Fwelch(3, 2.42)=20.9; p=0.029. Differences between groups young non-irradiated (2.360 ± 0.3796) and middle-aged non-irradiated (0.884 ± 0.1835; p=0.012) and middle-aged non-irradiated with middle-aged irradiated (2.125 ± 0.2141; p=0.022) are shown. D Absolute number of cells that had incorporated BrdU. This represents the relation between cell number counted after culture and the percentage of cells stained with anti-BrdU (Additional file 1: Fig. S3). One-way ANOVA F(3,6)=11.6; p=0.007; Differences between groups young non-irradiated (0.1403 ± 0.04) and middle-aged non-irradiated (0.0474 ± 0.01; p=0.008), young non-irradiated with middle-aged irradiated (0.0615 ± 0.003; p=0.018) and young non-irradiated with young irradiated (0.0398 ± 0.002; p=0.009) are shown. These graphics represent two independent experiments performed in triplicate. Red line represents the initial number of seeded cells (2.5x103). E Aged B-1p irradiated or aged non-irradiated samples were normalized with the respective sample of young mice and endogenous gene were ß2 microglobulin for Bcl-2 and Bax analysis or U6 for miR15a/16. Relative expression was evaluated by 2-ΔΔCt and here contains the ratio (fold change) between irradiated and non-irradiated group after 2-ΔΔCt calculus in each group (young or middle-aged). This graphic represents 2 experiments performed in triplicates