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. Author manuscript; available in PMC: 2020 Apr 16.
Published in final edited form as: Nature. 2019 Oct 16;574(7778):359–364. doi: 10.1038/s41586-019-1647-8

Extended Data Figure 7. Lifespan extension by spr-4 overexpression and inhibition of neural excitation are daf-16-dependent.

Extended Data Figure 7.

a, Lifespan extension by overexpression of spr-4 is daf-16 dependent. Lifespans of worms overexpressing spr-4 (sgRNA+;dCAS9::VP64) or not overexpressing spr-4 (sgRNA-;dCAS9::VP64) following treatment with daf-16 RNAi or an empty vector control. sgRNA(+)EV (n=29 worms) versus sgRNA(−)EV (n=25 worms): P=2.7e-4; sgRNA(+)daf-16 (n=18 worms) versus sgRNA(−)daf-16 (n=29 worms) P=0.20 by log-rank test. Representative of 4 independent replicates. b, c, Lifespan extension by the neural excitation inhibitors ivermectin and nemadipine is daf-16-dependent. Shown are lifespan determinations for WT control and daf-16(mu86) mutant worms in the presence or absence of nemadipine (2μM) (b) or ivermectin (1pg/ml) (c). b,WT, n=69 worms; WT+Nema, n=51; daf-16, n=43; daf-16+Nema, n=67. WT+Nema vs WT, P=9.9e-8; daf-16+Nema vs daf-16, P=0.014 by log-rank test c, WT, n=78 worms; WT+Ive, n=77; daf-16, n=27; daf-16+Ive, n=29. WT+Ive vs WT, P=7.3e-8; daf-16+Ive vs daf-16, P=0.22; log-rank test. Curves are representative of an experiment replicated 2 (nemadipine) or 3 (ivermectin) times. d, Inhibition of neural excitation with ivermectin elevates DAF-16 levels. Worms expressing a DAF-16::GFP transgene were treated for 10 days with 1 pg/mL ivermectin and assessed by confocal microscopy. Left panel: Shown is total DAF-16::GFP. Values represent the mean ± S.E.M (untreated, n=19 worms, Ivermectin, n=16 worms). **P=2.5e-7, Mann-Whitney U test. Right panel: Shown is nuclear DAF-16::GFP (n=5 worms per group, 50-61 nuclei per worm). *P= 0.013 by Student’s t-test. Results are representative of an experiment replicated twice. e, DAF-16 is not required for inhibition of neural excitation by nemadipine. Shown are maximum ASH GCaMP intensity changes for day 2 daf-16(mu86) mutant worms treated for 24 hours with 2 μM nemadipine (untreated, n=16 worms; nemadipine, n=18 worms). P=9.4e-5, Mann-Whitney U test). f, DAF-16 is not required for inhibition of neural excitation by ivermectin. Shown are day 2 worms treated for 24 hours with 1 pg/mL ivermectin (control, n=19 worms; ivermectin, n=32 worms). P=0.030, Mann-Whitney U test).