Etomoxir sodium salt

Oxygen consumption rate in wild-type worms and daf-11 mutants were measured using Seahorse XF96 extracellular flux analyzer (Agilent Technologies), following the protocol described in ref. ^{69 (link)}. Measurements were performed at room temperature (27 °C with heat generated by the machine). Age-synchronized worms were grown on E. coli OP50 NGM plates and collected as 1-day-old adults. Worms were washed in M9 buffer twice to remove bacteria, and approximately 25 worms were added in 200 μl M9 buffer to the wells of XF96 microplate. The oxygen consumption rate was measured five times at baseline and five times after the addition of etomoxir sodium salt (Sigma-Aldrich), an inhibitor of mitochondrial β-oxidation via carnitine palmitoyltransferase 1, dissolved in M9 to a 500 μM final concentration. For each well, the percent change in the average oxygen consumption rate before and after etomoxir addition was used to calculate the oxygen consumption rate dependent on mitochondrial fatty-acid β-oxidation. At least 10 wells were used for each wild-type and mutants in each assay and the assays were repeated two independent times. Worms in each well were counted after the assay is done and the oxygen consumption rate is normalized to worm number. Statistical analysis was performed using the Student’s t-test.