Lifespan assay

1. One hundred μl of OP50, which was cultured overnight at 37°C in liquid Luria broth (LB) media with 10 μg/ml streptomycin, was seeded on 35 mm NGM agar plates.
2. Animals were cultured at 20°C on nematode growth medium (NGM) agar plates seeded with Escherichia coli strain OP50.
3. Gravid worms were transferred to fresh OP50-seeded NGM plates, and kill them after 12-14 hours. 
4. When worms are pre-fertile adults, pre-fertile adult worms were transferred to fresh OP50-seeded NGM plate (30-35 worms/plate).
5. The worms were transferred again to fresh OP50-seeded plates every day until the worms stopped laying embryos.
6. The deaths of worms were scored every two days. Worms that did not respond to a gentle touch with a sterilized platinum wire were determined as dead.
7. Animals that burrowed, displayed internal hatching or vulval rupture, or crawled off the plates were censored but included in the statistical analysis.
8. Lifespan curves were generated and the statistical analysis was performed by using OASIS2 (https://sbi.postech.ac.kr/oasis2/) (Han et al., 2016). p-values were calculated using a log-rank (Mantel-Cox method) test. The lifespan curves were pooled from two or three independent lifespan data obtained by two researchers.

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