Expression of Drosophila MsrB in fruit flies. (A–D) Western blot analyses of fruit fly total homogenates with anti-Drosophila MsrB antibodies. dMsrB^2A (lanes 1, 2) and dMsrB^2B (lanes 3, 4) flies were crossed to GAL4-activator lines to overexpress Drosophila MsrB. Controls were obtained by crossing white flies with dMsrB^2A (lanes 5, 6), dMsrB^2B (lanes 7, 8) and GAL4-activator (lane 9) lines. F1 progeny of crosses between tubP-GAL4 (A), da-GAL4 (B), fatbody-GAL4 and elav-GAL4 (D) drivers and dMsrB (lanes 1–4) and white flies w¹¹¹⁸ (lane 9) were analyzed. Adult animals used were 6 days old. Migration of the 17 kDa band corresponding to fruit fly MsrB is shown by arrows on the left in panels A–D. (E) Protein loading control (42 kDa Drosophila β-actin) for the Western blotting experiment presented in A. Gender of 6-day-old females and males analyzed is shown by the corresponding symbols below panel E. (F) MsrB activity of total homogenate of fruit flies used for Western blot analyses. Designations 2A, 2B and letter w refer to mMsrB2^3A, mMsrB2^2A and w¹¹¹⁸ lines that were crossed with the indicated GAL4-driver. Measurements were performed in triplicate. All data are reported as the means ±S.D.

Survivorship curves of Drosophila MsrB-overexpressing flies on corn meal food. Males and females were kept together during lifespan study. Each survivorship curve represents 105 flies. Expression of Drosophila MsrB in whole body was driven by using tubP-GAL4 (A, E) or da-GAL4 (B, F) activator lines; in fatbody by using fatbody-GAL4 driver (C, G); in the nervous system by using elav-GAL4 line (D, H). Genotypes and sexes are shown on the plots. All trials were performed concurrently.

Analysis of Met and MetO content in 6- and 17-day-old flies. (A) Met content of the protein-free fraction of fruit fly homogenates. Met content is given in nmoles/mg of protein in homogenates. (B) Ratio of MetO to Met in a protein fraction. Flies (6- and 17- day-old) overexpressing mouse MsrB2 or Drosophila MsrB in whole body (da-GAL4/dMsrB and da-GAL4/mMsrB2), their corresponding driver line control da-GAL4/w¹¹¹⁸, and responder line controls w¹¹¹⁸/dMsrB and w¹¹¹⁸/mMsrB were homogenized and proteins were precipitated. The supernatant was used to determine protein-free Met (A), and the pellet was used for protein hydrolysis and analysis of MetO and Met content. 70 females were used for each sample. Two independent preparations of each transgenic fly line as well as control flies were analyzed. Measurements for each sample were done in triplicate. All data are reported as means ±S.D. The age of flies (in days) is shown for each column. Genotypes are indicated.

Age-associated changes in pupa production. Five females were kept with five males in a vial with corn meal food. Changes in pupa production were determined from counts of pupa developed from eggs laid by five female in 24 h; the developed pupa was counted after eclosion. The Y axis represents the number of pupa produced by one female in one vial. 10 replica of each vial were tested. Offsprings of crosses used (elav-GAL4/mMsrB2^3A, elav-GAL4/dMsrB^2A or elav-GAL4/w¹¹¹⁸) are shown in the legend.

Expression of mouse MsrB2 in fruit flies. (A–D) Western blot analyses of Drosophila total homogenates with anti-mouse MsrB2 antibodies. mMsrB2^3A (lanes 1, 2) and mMsrB2^2A (lanes 3, 4) lines were crossed to GAL4-activator lines to drive mouse MsrB2 expression. Control flies were obtained by crossing white flies (w¹¹¹⁸) with mMsrB2^3A (lanes 5, 6), mMsrB2^2A (lanes 7, 8) and GAL4-activator (lane 9) lines. Expression of mouse MsrB2 (lanes 1–4) in whole body was achieved by crossing mMsrB2 lines and tubP-GAL4 (A) and da-GAL4 (B) drivers, in fatbody by crossing mMsrB2 with fatbody-GAL4 driver (C), and in the nervous system by crossing mMsrB2 with elav-GAL4 driver (D). Migration of the 19 kDa band corresponding to mouse MsrB2 is indicated on the left in panels A–D. (E) Protein loading control (42 kDa Drosophila β-actin) for the Western blotting experiment shown in A. Gender of 6-day-old females and males analyzed is shown by the corresponding symbol below panel E. (F) MsrB activity of total homogenates of male flies used for western blot analyses. Designations 3A, 2A and letter w refer to mMsrB2^3A, mMsrB2^2A and w¹¹¹⁸ lines that were crossed with the GAL4-driver. Measurements of each sample were performed in triplicates. All data are reported as the means ±S.D.

Survivorship curves of mouse MsrB2 expressing flies on corn meal food. 35 female and 35 male animals were housed together in plastic cages; three replica cages were used for each lifespan curve. Survivorship distribution presented shows the average of those independent replicas (105 females and 105 males for each curve). Fresh corn meal food was supplied and surviving animals were counted every 3–4 days. Expression of mouse MsrB2 in whole body was activated by using tubP-GAL4 (A and E) or da-GAL4 (B and F) activator lines; in fatbody by using fatbody-GAL4 driver (C and G); in the nervous system by using elav-GAL4 line (D and H). Genotypes and sexes are indicated on the plots. All trials were performed concurrently.

Survivorship curves of mouse MsrB2- and Drosophila MsrB-overexpressing flies on sugar yeast food. (A) Survivorship curves of control flies (da-GAL4/w¹¹¹⁸) on normal and dietary restriction sugar yeast food (N SY and DR SY, respectively). (B–E) Survivorship of flies overexpressing mouse MsrB2 (B and C) and Drosophila MsrB (D and E) on DR SY food. Overexpression of MsrBs in whole body (B and D) and nervous system (C and E) was driven by da-GAL4 and elav-GAL4 activator lines, respectively. Males and females were kept together (A, C and E) or separate (B and D) during the lifespan study. 210 males were tested for each survivorship curve in panels B and D, and 105 animals for all other curves. Genotypes and sexes are marked on the plots. All trials were performed concurrently.

Stress resistance of mouse MsrB2- and Drosophila MsrB-overexpressing flies. 20-day-old male flies overexpressing MsrBs in the nervous system were examined for resistance to oxidative stress and starvation. Newly eclosed flies were mated, sex-segregated and maintained on corn meal food before the day of the tests. (A, B) Oxidative stress was induced by 20 mM (A) or 30 mM (B) dietary paraquat. (A) 80 animals were tested for each genotype (shown in the legend). Flies were transferred to test vials (5% sucrose, 20 mM paraquat) without preliminary starvation and dead animals were counted at fixed time points. 80 control flies (elav-GAL4/w¹¹¹⁸) were kept on 5 % sucrose without paraquat (marked as “no paraquat” in the legend). (B) 360 animals were tested for flies overexpressing mouse MsrB2 (elav-GAL4/mMsrB2), Drosophila MsrB (elav-GAL4/dMsrB), and control flies (MsrBs/w¹¹¹⁸), except for control elav-GAL4/w¹¹¹⁸ where 180 males were studied. Genotypes are shown in the Y-axis legend; 180 flies with genotype elav-GAL4/w¹¹¹⁸ were kept on food without paraquat and noted in Fig. 7B as “no paraquat”. All flies were starved for 6 h before the test and then fed with or without 30 mM paraquat and 5% sucrose for 17 h; animals alive were counted and normalized to the total number of animals used for each genotype. (C, D) Resistance to starvation was studied with flies expressing mouse MsrB2 (C) and Drosophila MsrB (D) in the nervous system. Flies were maintained in vials with 1% agar; dead animals were counted every 8 h. Total number of males tested for each genotype was 140. Genotypes are shown in the legends.