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# Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in *C. elegans*

Version of Record online: 23 FEB 2009

DOI: 10.1111/j.1474-9726.2009.00459.x

© 2009 The Authors. Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009

Additional Information

#### How to Cite

Greer, E. L. and Brunet, A. (2009), Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in *C. elegans*. Aging Cell, 8: 113–127. doi: 10.1111/j.1474-9726.2009.00459.x

#### Publication History

- Issue online: 26 MAR 2009
- Version of Record online: 23 FEB 2009
- Accepted for publication
*30 December 2008*

- Abstract
- Article
- References
- Supporting Information
- Cited By

**Figure S1** Addition of FUdR does not alter the dependency of sDR-induced lifespan extension on AMPK/*aak-2* or FoxO/*daf-16*. sDR extended WT (N2) worm lifespan (31.0% *P* < 0.0001) but did not significantly extend *aak-2*(*ok524*) mutant worm lifespan (2.3% *P* = 0.3295) or *daf-16*(*mu86*) mutant worm lifespan (4.3% *P* = 0.0624) on plates with 100 mg L^{−1} FUdR. Mean, standard errors, and statistical analysis for one experiment performed in triplicate are presented in Table S1C.

**Figure S2** AMPK and FoxO are not completely necessary for bDR lifespan extension. (A) In one experiment, bDR extended WT (N2) worm lifespan (101%, *P* < 0.0001), *aak-2*(*ok524*) mutant lifespan (47.3%, *P* < 0.0001), and *daf-16*(*mu86*) mutant lifespan (39.6%, *P* < 0.0001). Two-way anova revealed that the lifespan extension of WT (N2) worms across a bacterial gradient was significantly different from that of *aak-2*(*ok524*) mutant worms (*P* < 0.0001) or *daf-16*(*mu86*) mutant worms (*P* < 0.0001). (B) In a second experiment, bDR extended WT (N2) worm lifespan (59.1%, *P* < 0.0001), *aak-2*(*ok524*) mutant lifespan (27.4%, *P* < 0.0001), and *daf-16*(*mu86*) mutant lifespan (14.6%, *P* = 0.0120). Two-way ANOVA revealed that the lifespan extension of WT (N2) worms across a bacterial gradient was significantly different from that of *aak-2*(*ok524*) mutant worms (*P* < 0.0001) or *daf-16*(*mu86*) mutant worms (*P* < 0.0001). (C) In a third experiment, bDR extended WT (N2) worm lifespan (32.3%, *P* < 0.0001), *aak-2*(*ok524*) mutant lifespan (20.8%, *P* < 0.0001), and *daf-16*(*mu86*) mutant lifespan (26.7%, Two-way ANOVA revealed that the lifespan extension of WT (N2) worms across a bacterial gradient was not significantly different from that of *aak-2*(*ok524*) mutant worms (*P* = 0.1528) and *daf-16*(*mu86*) mutant worms (*P* = 0.6643). Mean, standard errors, and statistical analysis for these experiments performed in quadruplicates are presented in Table S3.

**Figure S3** FoxA/*pha-4* is not necessary for sDR to extend lifespan. (A) sDR extended the lifespan of WT (N2) worms treated with empty vector RNAi (E.V.) (34.7% *P* < 0.0001) or *pha-4* RNAi (36.7% *P* < 0.0001) initiated at L1, but not the lifespan of worms treated with *daf-16* RNAi (7.0% *P* = 0.1562). The efficacy of *pha-4* RNAi was confirmed by its ability to decrease worm fertility, consistent with its known role in pharyngeal organogenesis (Mango *et* *al*., 1994), and its effects on lifespan (Panowski *et* *al*., 2007). Mean, standard errors, and statistical analysis for two independent experiments performed in triplicate are presented in Table S7B. (B) sDR extended the lifespan of WT (N2) worms treated with empty vector RNAi (E.V.) (30.8% *P* < 0.0001) or *pha-4* RNAi (26.4% *P* < 0.0001) initiated at L4 but not the lifespan of worms treated with *daf-16* RNAi (6.1% *P* = 0.1801). Mean, standard errors, and statistical analysis for two independent experiments performed in triplicate are presented in Table S7B.

**Table S1** AMPK/aak-2 and FoxO/daf-16 are necessary for lifespan extension by sDR across a gradient of bacteria. (A) A serial dilution of bacteria extends WT (N2) worm lifespan but does not extend *aak-2(ok524)* mutant worm lifespan. Experiment #1 is displayed in Figure 1A. Combined p values were calculated using Fisher’s combined probability test. (B) A serial dilution of bacteria extends WT (N2) worm lifespan but does not extend *daf-16* (*mu86*) mutant worm lifespan. This experiment is displayed in Figure 1B. (C) Addition of FUdR does not alter the dependency of sDR-induced lifespan extension on AMPK/*aak-2* or FoxO/*daf-16*. This experiment is displayed in Figure S1. The mean lifespan values were calculated by a log-rank (Mantel-Cox) statistical test from triplicate samples of 30 worms each. *n*, number of observed dead worms/number of total worms.

**Table S2** Dilution of peptone (DP) extends lifespan in an AMPK/*aak-2* and FoxO/*daf-16* dependent manner. Experiment #2 is displayed in Figure 2A. The mean lifespan values were calculated by a log-rank (Mantel-Cox) statistical test from triplicate samples of 30 worms each. *n*, number of observed dead worms/number of total worms. Combined *P*-values were calculated using Fisher’s combined probability test.

**Table S3** bDR increase in worm lifespan is partially dependent on AMPK/*aak-2* and FoxO/*daf-16*. The average of these three experiments is displayed in Figure 2B. Each experiment is displayed in Figure S2. The mean lifespan values were calculated by a log-rank (Mantel-Cox) statistical test from quadruplicate samples of approximately 22 worms each. n, number of observed dead worms/number of total worms. Combined *P*-values were calculated using Fisher’s combined probability test.

**Table S4** (A) *eat-2(ad1116)* induced lifespan extension is independent of AMPK/*aak-2*. Experiment #1 is displayed in Figure 2C. Experiment #2 is displayed in Figure 6. (B) *eat-2(ad1116)* induced lifespan extension is independent of FoxO/*daf-16*. Experiment #2 is displayed in Figure 2D. The mean lifespan values were calculated by a log-rank (Mantel-Cox) statistical test from triplicate samples of 30 worms each. *n*, number of observed dead worms/number of total worms. Combined *P*-values were calculated using Fisher’s combined probability test.

**Table S5** Resveratrol increases worm lifespan in an AMPK/*aak-2* dependent but FoxO/*daf-16* independent manner.Experiment #2 is displayed in Figure 3A. Experiment #3 is displayed in Figure 3B. The mean lifespan values were calculated by a log-rank (Mantel-Cox) statistical test from triplicate samples of 30 worms each. *n*, number of observed dead worms/number of total worms. Combined *P*-values were calculated using Fisher’s combined probability test.

**Table S6** sDR increases worm lifespan in an AMPK/*aak-2* dependent and SIR2/*sir-2.1* independent manner. Experiment #3 is displayed in Figure 4A. The mean lifespan values were calculated by a log-rank (Mantel-Cox) statistical test from triplicate samples of 30 worms each. *n*, number of observed dead worms/number of total worms. Combined *P*-values were calculated using Fisher’s combined probability test.

**Table S7** sDR increases worm lifespan in a FoxA/*pha-4* independent manner. A) *smg-1* and *smg-1*; *pha-4* worms were grown at permissive temperature (24 °C) until the first day of adulthood when they were switched to 15 °C. Experiment #1 is displayed in Figure 4B. B) sDR extends the lifespan of WT(N2) worms treated with empty vector RNAi (E.V.) or *pha-4* RNAi bute not *daf-16* RNAi initiated at larval stage L1 or larval stage L4. L1 Experiment #1 is displayed in Figure S3A. L4 Experiment #2 is displayed in Figure S3B. The mean lifespan values were calculated by a log-rank (Mantel-Cox) statistical test from triplicate samples of 30 worms each. n, number of observed dead worms/number of total worms. Combined *P*-values were calculated using Fisher’s combined probability test.

**Table S8** sDR increases worm lifespan in an AMPK/*aak-2* dependent, *skn-1* independent manner. Experiment #2 is displayed in Figure 4C. The mean lifespan values were calculated by a log-rank (Mantel-Cox) statistical test from triplicate samples of 30 worms each. *n*, number of observed dead worms/number of total worms. Combined *P*-values were calculated using Fisher’s combined probability test.

**Table S9** sDR increases worm lifespan in an AMPK/*aak-2* and *clk-1* dependent, *hsf-1* independent manner. Experiment #2 is displayed in Figure 4C. Experiment #1 is displayed in Figure 5. The mean lifespan values were calculated by a log-rank (Mantel-Cox) statistical test from triplicate samples of 30 worms each. *n*: number of observed dead worms/number of total worms. Combined *P*-values were calculated using Fisher’s combined probability test. Note that experiment #2 of Table S9 was performed at the same time as experiment #2 of Table S8.

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Filename | Format | Size | Description |
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ACEL_459_sm_FigS1.eps | 103273K | Supporting info item | |

ACEL_459_sm_FigS2.eps | 104258K | Supporting info item | |

ACEL_459_sm_FigS3.eps | 104610K | Supporting info item | |

ACEL_459_sm_TablesS1-S9.pdf | 202K | Supporting info item |

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.