Fig. S1 RNAi-mediated knockdown of the 20S proteasome subunits β5 or α7 results in sharp decrease of proteasome activities. (B) Proteasome activity increases significantly in late larval and pupal developmental stages.

Fig. S2 Fluorescence units per μg of somatic tissue protein following measurement of the 26S (A1) and 20S (A2) proteasome peptidase activities or ROS levels (B) in tissue samples from young (A, B1) or aged (B2) flies treated for 6–9 days (A) or for 4 days (B) with the indicated doses of the PS-341 inhibitor (see also, Fig. 1A, 2E1, 2E3). Bars, ± SD (n = 3); *P < 0.05; **P < 0.01.

Fig. S3 (A) Fluorescence units per μg of somatic tissue protein following measurement of ROS levels in samples from young (Y), middle aged (M) and old (O) flies. (B) Differential sensitivity of young and aged flies to proteasome inhibition.

Fig. S4 Time-dependent effects of PS-341 on proteasome activities of young flies' somatic tissues.

Fig. S5 The ovarian and spermathecae tissues are relatively resistant (compared to somatic tissues) to proteasome inhibitors.

Fig. S6 Partial loss of proteasome function in the gonads induces a potent induction of proteasome genes and protein subunits in both the young and aged organism.

Fig. S7 Differential sensitivity of post-egg fertilization/laying developmental stages and gametogenesis to proteasome inhibition; for details of the experimental procedures, see Data S1 (Supporting information).

Fig. S8 Co-treatment of flies with proteasome inhibitors and a potent ROS scavenger (Tiron) suppressed proteasome-inhibition-mediated ROS accumulation; partially restored proteasome activities; and extended flies' lifespan.

Fig. S9 Proteasome inhibition in human cells triggers ROS accumulation and upregulation of the β5 proteasome subunit.

Fig. S10 (A) Relative (%) ROS level in flies' somatic tissues samples, after CncC knockdown in the absence or presence (for 3 days) of 5 μm PS-341. Bars, ± SD (n = 2); *P < 0.05. (B) CncC RNAi abolishes induction of proteasome subunits after proteasome loss of function independently of age.

Fig. S11 Transgene-mediated inducible CncC overexpression after flies' eclosion upregulates basal 19 and 20S proteasome gene expression, enhances proteasome activities, and mediates flies' resistance to stress; whereas in the long-term it shortens lifespan.

Fig. S12 RNAi-mediated Keap1 knockdown (via overexpression of an inverted repeat corresponding to the keap1-specific gene coding segment) enhanced proteasome genes expression and proteasome activities; conferred resistance to stress but in the long term reduced flies' lifespan.

Fig. S13 Inducible Nrf2 activation upregulates target proteasome genes in somatic tissues independently of age.

Fig. S14 A proposed model of proteasome activity-related feedback regulatory circuit.

acel12111-sup-0002-TableS1.pdfapplication/PDF106KTable S1 Statistics and presentation of lifespan experiments.
acel12111-sup-0003-DataS1.pdfapplication/PDF214KData S1 Experimental procedures, references and figure legends.
acel12111-sup-0004-VideoS1.avivideo/avi47212KVideo S1-S3 UAS B5/III control (video 1) and (III) UAS B5/tubGS5Gal4 F1 larvae (video 2) cultured in normal medium; (video 3) (III) UAS B5/tubGS5Gal4 F1 larvae cultured in RU486 containing medium.

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