Fig 5. nowl and Nf1 interact in the regulation of sleep.

(A) Daily sleep profiles of males under a 12-hour light/dark (white and black bars) cycle in 30-minute intervals. In elav>Nf1, nowl-RNAi animals (with neuronal overexpression of Nf1 and simultaneous knockdown of nowl), night-time sleep is partially restored compared to nowl knockdown alone. (B and C) Quantification of daytime (B) and nighttime (C) total sleep, sleep-bout duration, and sleep-bout numbers in males. The double knockdown (elav>nowl-RNAi, Nf1-RNAi) shows significant interactions between nowl and Nf1 in daytime total sleep and sleep-bout duration and number, indicating an additive effect in the daytime. The effects of double knockdown on total nighttime sleep and nighttime sleep-bout duration and number are not different from Nf1-RNAi alone, suggesting an epistatic relationship between nowl and Nf1 in night-time sleep regulation. Nf1 overexpression partially rescues the nowl-knockdown effect on sleep. Controls are elav>+. Additional effector line controls (nowl-RNAi/+, Nf1-RNAi/+, and Nf1/+) are shown in S8 Fig Genotypes that are indicated as different from the elav>+ controls are also statistically different (P< 0.05) from controls shown in S8 Fig (D) Daily sleep profiles of double-knockdown male animals (elav>Nf1-RNAi, nowl-RNAi) display a pronounced decrease in sleep. Controls are elav>+. (E) Western blotting against phosphorylated ERK (pERK) in males shows that single and double knockdowns of nowl and Nf1 cause increased pERK levels, standardized to alpha-Tubulin (controls are elav>+). Western blot is representative of two independent experiments. Graphs represent means with SEM (n = 32–81) of data pooled from one to three independent experiments. Significance was determined using Kruskal-Wallis test with Dunn's post-hoc testing (*p < 0.05, ** p < 0.01, *** p < 0.001).