Fig. S1 (A and B) act-1 quantitative PCR calibration for hsf-1 mRNA quantification (corresponding Fig.  1A). (C–F) Raw data of Fig.  1A.

Fig. S2 Worms expressing GFP under the hsp-16.2 promoter were either grown on control bacteria (EV) or hsf-1 RNAi for one generation and bleached.

Fig. S3 To test whether exposure to 25 °C induces the heat shock response, CL2070 worms that were grown to adulthood at 20 °C were exposed for 3 h to either 25 °C (lane 2), 33 °C (lane 3), or left at 20 °C (lane 1).

Fig. S4 (A) To examine how dcr-1 RNAi treatment affects lifespan, CF512 animals were let hatched on EV bacteria and transferred onto dcr-1 RNAi in 3-h intervals from 3 to 18 or 36 h after hatching and their lifespans were recorded. Our results show that dcr-1 RNAi shortens lifespan when applied during larval development but this effect becomes less prominent when the worms had natural dicer levels for longer time from hatching. (B) Similarly we tested whether the knockdown of dcr-1 during adulthood modifies lifespans of CF512 worms. No effects on lifespan were observed when dcr-1 RNAi was applied at either days 1, 5, or 9 of adulthood.

Fig. S5 (A and B) Quantitative PCR indicates that the hsf-1 expression levels are efficiently decreased (70–80% compared to control) in wild-type larvae within 4–6 h after exposure to hsf-1 RNAi in L2 (A) and L4 (B) stages. (C and D) The expression levels of hsf-1 are restored to nearly natural levels in L2 larvae ~6 h after transfer onto dcr-1 RNAi (C) while in L4 larvae hsf-1 restoration is apparent 1.5 h after exposure to dcr-1 RNAi (D).

Fig. S6 Worms transferred from hsf-1 onto dcr-1 RNAi later than 12 h after hatching exhibited progressively shorter lifespans (corresponding Fig.  2E, corresponding Table  S5).

Fig. S7 (A) daf-2(e1370) mutant, but neither wild-type nor daf-2(e1368) mutant worm are developmentally arrested when grown for two generation on hsf-1 RNAi (white arrows point to progeny). (B) daf-2(e1368) grown for three generations on hsf-1 RNAi completed development. (C) Experimental design of Fig.  3A,B.

Fig. S8CF512 worms were grown on EV bacteria up to days 1, 4, or 8 of adulthood and either exposed for 24 h to hsf-1 RNAi or left untreated prior to exposure to heat shock (33 °C, 1 h).

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