RNAi was performed via feeding of in vitro–synthesized double-stranded RNAi, as previously described (35). A 489 base pair (bp) region of S. mediterranea SOD (SMU15011417) was used to generate SOD RNAi. The primers were 5′-ACTGGAGCCATCAATATCTGG and 3′-TAATCCGGCCTTACATTTTTG. A 552 bp region of S. mediterranea Hsp70 (SMU15039086) was used to generate Hsp70 RNAi. The region was from 5′-GGTTTTTGATTTGGGTGGTG to 3′-AGCTGTTGCTATGGGAGC. Worms were fed with RNAi three times over 8 days before being amputated on day 9, as indicated above. Control RNAi was double-stranded RNA to Venus-GFP, which is not present in the planarian genome. SOD RNAi rescue experiments were performed twice (except for 45 μT + SOD RNAi). Total biological replicates for each condition were as follows: 45 μT, n = 20; 200 μT, n = 20; 45 μT + SOD RNAi, n = 10; and 200 μT + SOD RNAi, n = 20. Total biological replicates for each condition for Hsp70 RNAi morphology experiments were as follows: control RNAi, n = 15 and Hsp70 RNAi, n = 15.

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