Research Paper Volume 9, Issue 4 pp 1153—1185

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.