Effects of CncC and dKeap1 depletion on the time of pupation, pupal size, and ecdysteroid levels.

<p>(A). Effects of expression of the shRNA targeting CncC in the PG on the time of pupation. The proportion of larvae that had formed pupae was plotted as a function of the time after 3^rd instar ecdysis for control larvae (<i>phm></i> or <i>5015></i>, black; <i>cncC-RNAi</i>, purple), for larvae that expressed the shRNA targeting CncC (<i>phm>cncC-RNAi</i>, green; <i>5015>cncC-RNAi</i>, dark green), as well as for larvae that expressed a different shRNA targeting all Cnc isoforms (<i>phm>cnc-RNAi</i>, cyan) in the PG. Pupation by the larvae the expressed the shRNA targeting CncC was also examined when the food was supplemented with 0.5 mg/ml 20E (<i>phm>cncC-RNAi</i>+20E, red). The data in the different graphs were obtained from separate experiments and represent the means and standard deviations from two to four repeats using 20–30 larvae in each. (B). Effects of expression of the shRNA targeting CncC in PG on the sizes of pupae. The lengths of the pupae formed by control larvae (<i>phm></i>) and larvae that expressed the shRNA targeting CncC in the PG (<i>phm>cncC-RNAi</i>) were measured. The data represent the mean and standard deviation of 30 pupae of each genotype (*, p<0.001). (C). Effects of expression of the shRNA targeting CncC in the PG on 20-hydroxyecdysone (20E) levels. The levels of 20E in larvae and white pre-pupae (WPP, open symbols) were measured in control (<i>phm></i>, black) and transgenic <i>Drosophila</i> that expressed the shRNA targeting CncC in the PG (<i>phm>cncC-RNAi</i>, green). The 20E level at each time point was measured in 10 larvae or pre-pupae. (D). Effects of expression of the shRNA targeting dKeap1 in the PG on the time of pupation. The proportion of larvae that had formed pupae was plotted as a function of the time after hatching for control larvae (<i>phm></i>, black) and for larvae that expressed the shRNA targeting dKeap1 (<i>phm>dKeap1-RNAi</i>, orange) in the PG. The data represent the means and standard deviations from four separate experiments using 20–30 larvae in each. The time of pupation was measured after hatching since it was more difficult to obtain a sufficient number of larvae synchronized at 3^rd instar ecdysis. Based on observation of a small number of larvae (>10), the time between 3^rd instar ecdysis and pupation was delayed by about 3 days upon dKeap1 depletion in the PG. The delay in pupation was therefore primarily due to extension of the 3^rd instar larval stage. (E). Effects of expression of the shRNA targeting dKeap1 in the PG on the sizes of pupae. The lengths of the pupae formed by control larvae (<i>phm></i>) and larvae that expressed the shRNA targeting dKeap1 in the PG (<i>phm>dKeap1-RNAi</i>) were measured. The data represent the mean and standard deviation of 20 pupae of each genotype (*, p<0.001).