Fig. 1

Outgrowth assays determined the anti-aging activity of 2,5-AM and rapamycin in human cells. a CLS determination by qualitatively assessing the cells’ ability to proliferate. Cells were plated without (control) or with 2,5-AM compound treatment at several concentrations. On the fourth day after cells were seeded and treated, cells were trypsinized and 2% (4 µl out of 200 µl) of the cells were transferred to a 6-well experiment plate with fresh D10 medium. The experiment plate was incubated at 37 °C incubator with 5% CO₂ for seven days before staining with the Crystal Violet Assay. b CLS determination by quantitatively assessing the cells’ ability to proliferate with 2,5-AM treatment. The same cells were used as in (a), 10% (20 µl out of 200 µl) of the trypsinized cells were transferred to a 96-well experiment. c CLS determination by the outgrowth assay with rapamycin treatment. A similar experiment procedure was done as in (b) with 2,5-AM. d Schematic diagram illustrating the experimental procedure of the outgrowth assay. b–c were analyzed by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons tests. Results are plotted as mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001