Figure 6. CM from aged callus inhibits the growth of MPCs, which is prevented by senolytic drugs.

Young and aged mice were sacrificed on 10 dpf. (A) Callus pieces were cultured for 2 days to generate CM. CaMPCs were treated with 30% of CM for 2 days and subjected to growth, proliferation, apoptosis, and senescence analyses. (B) Cell growth, as in Figure 5E. n = 4 wells. The experiment was repeated twice. (C) Cell proliferation was assessed according to the percentage of cells incorporating BrdU. BrdU⁺ cells are indicated by white arrowheads. Scale bars: 100 mm. Original magnification, ×4 (enlarged insets). n = 4 wells. The experiment was repeated once. (D) Cell apoptosis was measured by flow cytometry as the percentage of annexin V⁺ cells. The experiment was repeated once. (E) Cellular was senescence assessed according to the percentage of SA–β-gal⁺ cells. n = 4 wells. The experiment was repeated once. (F) The expression of senescence markers was determined by qPCR. n = 3 wells. Relative mRNA expression is the fold change versus young cells as 1. The experiment was repeated once. *P < 0.05, by unpaired, 2-tailed Student’s t test (B, C, E, and F). (G) CaMPCs were treated with CM with or without 200 nM dasatinib plus 20 μM quercetin. n = 4 wells. The experiment was repeated once. *P < 0.05, for vehicle versus D+Q, by 2-way ANOVA followed by Tukey’s post hoc test. Only comparisons between vehicle versus D+Q treatment in young or aged mice are shown. ctl, control.