Volume 27, Issue 1, Pages 1-15 (January 2017) The Mitochondrial Lon Protease Is Required for Age-Specific and Sex-Specific Adaptation to Oxidative Stress  Laura C.D. Pomatto, Caroline Carney, Brenda Shen, Sarah Wong, Kelly Halaszynski, Matthew P. Salomon, Kelvin J.A. Davies, John Tower  Current Biology  Volume 27, Issue 1, Pages 1-15 (January 2017) DOI: 10.1016/j.cub.2016.10.044 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2017 27, 1-15DOI: (10.1016/j.cub.2016.10.044) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 H2O2 Pretreatment Induces Lon in a Female-Specific Manner that Diminishes with Age (A–F) Control flies assayed for lon mRNA and protein following H2O2 pretreatment. (A and B) lon mRNA in (A) 3- and 60-day females and (B) 3- and 60-day males. (C) Lon protein increased upon various concentrations of H2O2 in 3-day females. (D) Lon protein unchanged in 3-day, pretreated males. (E) Lon protein not inducible upon H2O2 pretreatment in 60-day females. (F) Lon protein unchanged in 60-day, pretreated males. Western blots were performed in triplicate, normalized to anti-Actin-horseradish peroxidase (HRP) and quantified using ImageJ. Quantification is a 100-kDa band, additional band (60 and 50 kDa) quantification is presented in Figures S1C and S1D. (G and H) Proteolytic capacity to degrade oxidized (tritiated [3H]) aconitase in presence of 5 mM ATP measured in mitochondria from 3- and 60-day control flies following H2O2 pretreatment. (G) Females. (H) Males. Samples were normalized to 3- and 60-day 0-μM controls. Statistical significance (p < 0.05) calculated here and below using one-way ANOVA is indicated by asterisk (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). See also Figures S1 and S2. Current Biology 2017 27, 1-15DOI: (10.1016/j.cub.2016.10.044) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Lon Is Required for H2O2 Adaptation in Females Actin-GS-255B was crossed to Lon R1 (A, B, E, and F) or Lon R2 (C, D, G, and H) to knock down Lon expression. All progeny were cultured with or without RU486 for 9 days prior to pretreatment. At day 10, flies were cultured in presence or absence of adaptive dose of H2O2 before being fed H2O2 challenge dose (4.4 M). (A and C) Females without RU486. (B and D) Females with RU486. (E and G) Males without RU486. (F and H) Males with RU486. Statistically significant difference in survival (p < 0.05) is calculated here and below using log-rank test and indicated by p value and percentage change median. Statistical summary is in Table S3. See also Figures S1–S6 and Tables S2 and S4. Current Biology 2017 27, 1-15DOI: (10.1016/j.cub.2016.10.044) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Equal Sex Sensitivity to PQ but Only Males Adapt (A–D) Survival for control flies with increasing concentrations PQ. (A) 3-day females. (B) 3-day males. (C) 35-day females. (D) 35-day males. (E–H) Survival for control flies fed adaptive doses PQ (0–10 μM) prior to challenge dose (30 mM). (E) 3-day females. (F) 3-day males. (G) 35-day females. (H) 35-day males. Statistical summary is in Table S1. See also Figure S2 and Table S6. Current Biology 2017 27, 1-15DOI: (10.1016/j.cub.2016.10.044) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 PQ Pretreatment Induces Lon in a Male-Specific Manner that Diminishes with Age (A and B) Control flies assayed for lon mRNA expression following PQ pretreatment. lon mRNA in (A) 3- and 60-day females and (B) 3- and 60-day males. (C–F) Lon protein following PQ pretreatment. Western blots were performed in triplicate and quantified using ImageJ. (C) 3-day females. (D) 3-day males. (E) 60-day females. (F) 60-day males. (G and H) Proteolytic capacity to degrade oxidized [3H] aconitase in presence of 5 mM ATP measured in mitochondria from 3- and 60-day control flies following PQ pretreatment. (G) Females. (H) Males. Current Biology 2017 27, 1-15DOI: (10.1016/j.cub.2016.10.044) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Lon Is Required for PQ Adaptation in Males Lon R1 and Lon R2 males were crossed to virgin females of Actin-GS-255B, and progeny was cultured with or without RU486 for 9 days prior to pretreatment. Statistical summary is in Table S5. See also Figures S2–S4 and S6. (A and C) Females without RU486. (B and D) Females with RU486. (E and G) Males without RU486. (F and H) Males with RU486. Current Biology 2017 27, 1-15DOI: (10.1016/j.cub.2016.10.044) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 The Female Lon Banding Pattern Is Recapitulated in Pseudo-females (A) UAS-TraF × Actin-GS-255B Gene-Switch pseudo-female formation. RU486 activates TraF in males, producing female morphology (“pseudo-females”). (B–G) UAS-TraF crossed to Actin-GS-255B, with resulting larvae cultured in either 160 (1×) or 320 μg/mL (2×) RU486. (B) qPCR analysis of transcript variants lon RA and lon RC from progeny cultured in 2× ± RU486. (C) Western blot using anti-Lon antibody. (D) Male sex comb. Upon increasing RU486, the sex comb shrinks (1×) until no longer present (2×) RU486. (E) Whole flies. (F) Western blot of whole fly, dissected gonads, and carcass minus gonads. (G) Male genitalia in progeny grown without RU486. Formation of female genitalia in pseudo-females grown in (2×) RU486. (H–J) Proteolytic capacity of pretreated males without RU486, 2× RU486 pseudo-females, and 2× ± RU486 females to degrade [3H] aconitase (H) H2O2 pretreatment. (I) PQ pretreatment. (J) H2O2 pretreatment in RU486-fed adults. (K) UAS-Tra RNAi crossed to Actin-GS-255B, larvae cultured in 2× RU486. Proteolytic activity assayed following H2O2 pretreatment. See also Figure S7. Current Biology 2017 27, 1-15DOI: (10.1016/j.cub.2016.10.044) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Adaptation in Transformed Flies and Sex-Biased Lon Isoform Expression across Species (A–H) UAS-TraF crossed to Actin-GS-255B, resulting larvae cultured in 2× RU486. (A–D) Survival following H2O2 pretreatment. (A) Males raised without RU486. (B) Pseudo-females. (C and D) Female progeny cultured (C) without RU486 and (D) with 2× RU486 prior to H2O2 pretreatment. (E–H) Survival following PQ pretreatment. (E) Males raised without RU486. (F) Pseudo-females. (G and H) Females were cultured (G) without RU486 or (H) with 2× RU486. (I–L) UAS-TraF crossed to Actin-GS-255B and adult progeny cultured ± 2× RU486 prior to H2O2 pretreatment. Males without RU486 (I) or with RU486 (J). Females without RU486 (K) or with RU486 (L). (M–P) Survival following H2O2 pretreatment in progeny of Tra RNAi crossed to Actin-GS-255B. (M) Males raised without RU486. (N) Males raised with RU486. (O) Females raised without RU486. (P) Pseudo-males raised without RU486. Statistical summary is in Table S7. (Q–T) Western blots using mouse anti-Lon antibody in female (F) and male (M) tissue from 3-month black C57BL/6 strain. Anti-actin-HRP loading control. (Q) Hindleg. (R) Heart. (S) Liver. (T) Ovaries and testes. See also Figure S7. Current Biology 2017 27, 1-15DOI: (10.1016/j.cub.2016.10.044) Copyright © 2017 Elsevier Ltd Terms and Conditions