ATP Assay for Drosophila Samples

ATP assays were conducted as described (Pogson et al., 2014 (link)) with some modifications. Briefly, ten heads of 3- to 5-day-old male flies were homogenized in 100 µl extraction buffer (6 M guanidine-HCl, 100 mM Tris, 4 mM EDTA, pH 7.8). After homogenization, samples were frozen in liquid nitrogen, followed by boiling for 5 min. The samples were centrifuged at 18,400 g for 3 min at 4°C, and supernatants were diluted (1:10) with extraction buffer and mixed with a luminescent solution (CellTiter-Glo Luminescent Cell Viability Assay, Promega, Fitchburg, WI, USA). Luminescence was measured on a Veritas™ Microplate Luminometer (Promega, Fitchburg, WI, USA). Relative ATP levels were calculated by dividing the luminescence by the concentration of the control. The relative ATP levels of each group were statistically analyzed by Tukey–Kramer test.