Extended Data Fig. 7: Senescence of aged neuroblasts increases NK cell number and activity.
Single-cell suspensions were prepared from dentate gyrus of male young (3 months) and aged (18 months) DCX-mRFP reporter mice. Thereafter, mRFP+ neuroblasts were sorted using flow cytometry and cultured in vitro. a, Real-time PCR and western blotting assessment of senescence markers (P16, P21 and P53) at mRNA and protein levels in cultured neuroblasts receiving treatment with mTOR inhibitor rapamycin or vehicle for 48 h. *p<0.05, **p<0.01 by two-tailed unpaired Student’s t-test. Left: n = 5 per group. P16: p=0.006, t=3.705, df=8; P21: p=0.0237, t=2.786, df=8; P53: p=0.0007, t=5.324, df=8. Right: n = 3 per group. P16: p=0.0324, t=3.217, df=4; P53: p=0.0073, t=5.037, df=4. b, Flow cytometry analysis of senescence-associated β-galactosidase (β-gal) expression in groups of cultured young (3 months) and aged (18 months) neuroblasts receiving indicated treatment for 48 h. n = 4 per group. **p<0.01 by one-way ANOVA. F2,9=20.01. Aged + vehicle vs. aged + rapamycin, p=0.002, q=7.012, df=9; aged + vehicle vs. aged + mTOR siRNA, p=0.0006, q=8.319, df=9. c, Flow cytometry analysis of IL-27 expression in groups of cultured young (3 months) and aged (18 months) neuroblasts receiving indicated treatment for 48 h. n = 8 per group. **p< 0.01 by one-way ANOVA. F2,21=11.0. Aged + vehicle vs. aged + rapamycin, p=0.0022, q=5.525, df=21; aged + vehicle vs. aged + mTOR siRNA, p=0.0011, q=5.976, df=21. d, Flow cytometry analysis of expression of NKG2D or Granzyme B in groups of NK cells after coculture with aged (18 months) neuroblasts receiving indicated treatment for 48 h. n = 8 per group. **p<0.01 by one-way ANOVA. NKG2D: F3,28=15.22. Aged + vehicle vs. aged + rapamycin, p=0.0045, q=5.276, df=28; aged + vehicle vs. aged + mTOR siRNA, p<0.001, q=7.413, df=28; aged + vehicle vs. aged + IL-27 siRNA, p<0.001, q=8.927, df=28. Granzyme B: F3,28=8.171. Aged + vehicle vs. aged + rapamycin, p=0.0094, q=4.865, df=28; aged + vehicle vs. aged + mTOR siRNA, p=0.0015, q=5.873, df=28; aged + vehicle vs. aged + IL-27 siRNA, p=0.001, q=6.106, df=28. Data are representative of three independent experiments. Error bars represent s.e.m.
