Fig. 4

Exogenous EVs from senescent NSCs activate the p53 pathway. A. Treatment with P3 and P5 EVs^WT diminished proliferation in the recipient P3 NSCs^WT. Cell proliferation was visualized by a) immunostaining with Ki67 (Red) and Nestin (Green). Scale bar: 20 μm. b) quantification of Ki-67 positive cell count. Two-way ANOVA followed by Sidak’s multiple comparisons test was performed. B. and C. Expression of P16 and P53 detected by Western blot in P3 NSCs^WT treated with P3 or P5 EVs^WT for 24 h. Full-length gels are presented in Additional file 1: Figs. 4B and 4C. a) gel images; b) densitometry. One-way ANOVA followed by Bonferroni’s Multiple Comparison Post-Test was performed. D. a) Removal of misfolded SOD1 from P5 EVs^WT restored the proliferation of NSCs. b) Incubation of GST-tagged CT4 and P5 EVs^WT for 2 h resulted in a pulldown of a misfolded SOD1 band as detected by Western blot. After removing misfolded SOD1, P5 EVs^WT lost its inhibition on the proliferation of P3 NSCs^WT as determined by immunostaining with antibodies to Ki67 (Red) and Nestin (Green). Scale bar: 20 μm. c) For quantitative comparison of Ki-67 positive cells, a Student’s t-test was performed. The experiments were done in triplicate from three independent cultures