CD49f⁺cells show an aging-related increase in their 3GF concentration requirement to stimulate their division. (A) Weighted 3GF dose-response curves for CD49f⁺ cell survival (top) and proliferation (bottom) monitored over a 7-day period in vitro as described in Figure 3A. Proliferation curves are normalized to cells that survived either past a first division or for 7 days without dividing. Points indicate the mean ± 1 SEM of the survival and proliferation responses of each group of cell types assessed (∗P < .05; ∗∗∗P < .001). The 50% effective dose concentrations were interpolated from the fitted curves (survival: CB = 0.11%, young adult = 0.09%, aged adult = 0.19%; proliferation: CB = 0.2%, young adult = 0.6%, aged adult = 0.9%). (B) Percent of clones that completed the indicated number of divisions by day 7 at 100% 3GF (left) or 1% 3GF (right). Lines connect the observed mean ± 1 SEM of the proportion of cells from each donor source that had completed the indicated number of divisions within 7 days. (∗∗∗P < .001; pairwise Kruskal-Wallis tests). (C) Kinetics of 3GF-stimulated timing of a first division of CD49f⁺ cells from different sources exposed to 100% (solid lines) or 1% (dotted lines) 3GF cocktail (CB: 100% and 1% 3GF, 57 hours and 72 hours, respectively; young adult: 100% and 1% 3GF, 77 hours and 116 hours, respectively; aged adult: 100% and 1% 3GF, 85 hours and 145 hours, respectively). Error bars drawn at the median value of each curve indicate the range defined by ± 1 SEM of this estimate for each sample type. (D) Proportion of CD49f⁺ cells cultured in 1% 3GF in different phases of the cell cycle assessed at different times. The size of each circle represents the proportion of total cells in each phase at the time shown (∗P < .05; ∗∗P < .01; ∗∗∗P < .001; Holm-corrected Wilcox tests). (E) Scaled intensity (Z scores) of CDK6, CDK2, Ki67, and pRb protein levels found in the CD49f⁺ cells analyzed (∗P < .05; ∗∗P < .01; ∗∗∗P < .001; Holm-corrected Wilcox tests).