Research Paper Volume 9, Issue 4 pp 1153—1185

The age- and sex-specific decline of the 20s proteasome and the Nrf2/CncC signal transduction pathway in adaption and resistance to oxidative stress in Drosophila melanogaster

Figure 9. Adaptation is dependent upon the 20S proteasome. Progeny of the Actin-GS-255B strain crossed to the β1 or β2 RNAi strains were aged for 5 days in the absence or presence of RU486 prior to H2O2 pretreatment. (A-D). The purpose of the experiment was not to completely knockdown the entire pool of 20S proteasome, but only to block the transcription/translation-dependent adaptive increase in proteasome expression following hydrogen peroxide pretreatment. Thus, we used RNAi conditions that blocked increased proteasome expression, without depressing basal proteasome protein levels. Using this approach, we found at least a 50% decrease in mRNA in RNAi strains, and within proteasome western blots and activity, we found blockage of the adaptive increase. After pretreatment, proteolytic capacity of the individual subunits of the 20S proteasome (trypsin-like, caspase/peptidyl glutamyl-peptide hydrolyzing-like activity, and chymotrypsin-like activity) were measured in whole fly lysate. (A-B) Proteolytic capacity in β1 RNAi flies in the absence (black) “control” or presence (pink in females or blue in males, denoted with “+RU486”) of RU486. (A) Females. (B) Males. (C-D) Proteolytic capacity in β2 RNAi flies in the absence (black) “control” or presence (pink in females or blue in males, denoted with “+RU486”) of RU486. (C) Females. (D) Males. (E,G) Females of the β1 and β2 RNAi strains raised in the absence of RU486 were either not pretreated “control” (black circle) or were pretreated with either 10µM H2O2 (grey squares) or 100µM H2O2 (grey circles) for 8 hours, followed by a 16-hour recovery prior to H2O2 [4.4M] challenge. Females of the β1 and β2 RNAi strains raised in the presence of RU486 were either not pretreated “+RU486” (pink triangle) or were pretreated with either 10µM H2O2 (pink diamonds) or 100µM H2O2 (pink squares) for 8 hours, followed by a 16-hour recovery prior to H2O2 [4.4M] challenge. (F,H) Males of the β1 and β2 RNAi strains raised in the absence of RU486 were either not pretreated “control” (black circle) or were pretreated with either 10µM H2O2 (grey circles) or 100µM H2O2 (grey triangles) for 8 hours, followed by a 16-hour recovery prior to H2O2 [4.4M] challenge. Males of the β1 and β2 RNAi strains raised in the presence of RU486 were either not pretreated “+RU486” (green circle) or were pretreated with either 10µM H2O2 (blue square) or 100µM H2O2 (blue circle) for 8 hours, followed by a 16-hour recovery prior to H2O2 [4.4M] challenge. Statistical difference in survival (p < 0.05) was calculated using the Log-Rank test. Statistical summary is located in Supplementary Table S2.