Fig 1. ATP and p-AMPK levels fall during DTS and SIS.

(A and C) Total body ATP normalized by μg protein (A) and total body p-AMPK normalized to actin loading control (C) in WT animals before, during, and after lethargus/DTS of the L1 stage. Shown is a representative time course performed in technical duplicates of an experiment that was replicated 6 times for ATP levels (the average of the 7 biological replications is shown in Fig 3F) and 3 times for p-AMPK staining (Fig 3G). p-AMPK and ATP levels were measured from worms grown in the same batch. Data are represented as the mean ± SD with 2 technical replicates for ATP and AMPK. The second y-axis shows the averaged fraction of nonpumping animals (n = 10) for each time point. Statistical comparisons were performed with a 2-way ANOVA, followed by post hoc pairwise comparisons at each time point to obtain nominal p-values, which were subjected to a Tukey multiple-comparison correction. ATP values that are significantly different from the largest value in the time course are indicated by *** (p < 0.001), ** (p < 0.01), and * (p < 0.05) (S1 Data, Sheet 1A and 1C). A representative western blot is shown below the graph, in which the intensities of the upper bands represent levels of p-AMPK using an antibody against mammalian p-AMPKα Thr-172, and the intensities of the lower bands represent levels of the β-actin loading control. (B and D) Total body ATP normalized by μg protein (B) and total body p-AMPK normalized to actin loading control (D) as well as movement quiescence in WT adults after UVC irradiation. We measured body movement quiescence of animals that were in the same batch used for ATP measurements. Shown is a representative time course of an experiment run independently 6 times for ATP (Fig 3H) and 3 times for p-AMPK staining (Fig 3I). Data are represented as the mean ± SD with 3 technical replicates for ATP. For p-AMPK, data are represented as the mean ± SEM of 2 experiments. The second y-axis shows the averaged movement quiescence of animals (n = 5–7) for each time point. Statistical comparisons were performed with a 2-way ANOVA, followed by post hoc pairwise comparisons at each time point to obtain nominal p-values, which were subjected to a Tukey multiple-comparison correction. *** and * indicate corrected p-values that are different from the 0-hr time point at p < 0.001 and p < 0.05, respectively (S1 Data, Sheet 1B and 1D). A representative western blot of p-AMPK and actin levels is shown below the graph. (E) Schematic of a histamine-mediated chemogenetic inhibition experiment to deprive worms of SIS. Age-matched worms were grown from the L1 stage to adulthood in the absence of histamine. One-day-old adults were transferred to individual wells of a WorMotel device with each well containing 10 mM histamine or vehicle control agar. Approximately 15 min after transfer, worms were exposed to UVC irradiation (1,500 J/m²), and their body movement quiescence was recorded for 8 hr. See also Material and methods. (F) Minutes of body movement quiescence during the first 4 hr after UVC exposure/SIS in adult animals expressing either the Pflp-11::HisCl transgene (+) or in nontransgenic animals (-). Shown is the combined data of 2 biological replicates as shown in S2B Fig. Data are represented as mean ± SEM. ***p < 0.001 by a 2-tailed Mann-Whitney t test (S1 Data, Sheet 1F). (G) Total body ATP per μg protein measured 0 and 2 hr (during maximal movement quiescence) after UVC exposure in adult animals expressing either the Pflp-11::HisCl transgene or in nontransgenic animals in the presence of 10 mM histamine (+His) compared with WT adults in the absence of histamine (-His). Data were normalized to WT controls immediately before UVC exposure (0 hr) in the absence of histamine (-His). The graph shows the mean ± SEM of 3 experiments. *** indicates values that are different from that of nontransgenic animals (+His) at p < 0.001, and ns indicates p > 0.05. Statistical comparisons were performed with a 2-way ANOVA using time and genotype as factors, followed by post hoc pairwise comparisons at each time point to obtain nominal p-values, which were subjected to a Tukey correction for multiple comparisons (S1 Data, Sheet 1G). AMPK, adenosine monophosphate regulated protein kinase; DTS, developmentally timed sleep; L1 stage, first larval stage; ns, not significant; p-AMPK, phosphorylated AMPK; SIS, stress-induced sleep; UVC, ultraviolet C; WT, wild type.