1. Reversible Age-Related Phenotypes Induced during Larval Quiescence in C. elegans 
Antoine E. Roux, Kelley Langhans, Walter Huynh, Cynthia Kenyon 
Cell Metabolism 
Volume 23, Issue 6, Pages (June 2016)
DOI: /j.cmet
Copyright © Terms and Conditions

2. Cell Metabolism 2016 23, 1113-1126DOI: (10.1016/j.cmet.2016.05.024)
Copyright © Terms and Conditions

3. Figure 1
Appearance of Age-Related Phenotypes during C. elegans L1 Developmental Arrest
(A) Timeline depicting the experimental protocol.
(B) Lifespans of adults previously L1 arrested for 1 or 17 days as L1s were the same. n(blue) = 74 and n(red) = 88; p = 0.95 (see Table S1).
(C) Nomarski images after 1 day or 30 days of C. elegans L1 arrest. Scale bar, 10 μm.
(D) Survival declines during L1 arrest. Animals first lose the ability to develop when fed, and subsequently, as assayed by their failure to move when prodded, they die. Ratio comparing developing ability relative to survival, and replicates are shown in Table S2.
(E) Representative confocal images showing fragmentation of the mitochondrial network in muscle cells of aging adults and arrested L1s. Mitochondria were visualized using a mitochondrial-targeted GFP protein (Pmyo-3::mito-gfp). The strain also expressed nuclear-localized GFP (Pmyo-3::NLS-gfp) to facilitate cell identification. Scale bar, 10 μm. n, nucleus; M, mitochondria. Quantifications and additional pictures are shown in Figures S1C, S1D, S1F, and S1G.
(F) Increased ROS levels in aging adults and arrested L1s, visualized by microscopy using DHE (quantification in Figure S3A). Scale bar, 10 μm. G, intestinal granules, also labeled with DHE.
(G) DTT resistance declined during L1 arrest. The graph shows the percentage of L1s that developed after stress, normalized to the percentage alive before stress. n > 100 in each of three independent cultures, ∗∗p = Error bars represent SDs.
(H) Heat resistance declined during L1 arrest. The graph shows the percentage of L1s that developed after stress relative to the percentage of L1s that developed without stress. n > 100 in three independent cultures, ∗p = Error bars represent SDs.
(I) Increased KIN-19::tagRFP aggregation in aging adults and arrested L1s. Scale bar, 10 μm (quantification in Figure S3C).
(J and K) Muscle-specific polyglutamine (Pmyo-3::polyQ35::yfp) aggregated in aging adults (J) and arrested L1s (K). Each dot represents a worm. L2, L3, and L4, larval stages. Blue bar, average (pictures in Figure S3E).
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © Terms and Conditions

4. Figure 2
Age-Related Phenotypes, but Not Residual Fat Content, Predict the Ability of Arrested L1s to Recover when Fed
(A) The abundance of fat granules in L1s arrested 20–21 days, measured with Bodipy-C12 fluorescent dye in singled animals, did not correlate with the ability, or the time, to develop to the L2/L3 stage after feeding. a.u., arbitrary unit of green fluorescence intensity measured in the entire L1 (p = 0.97) (supporting data, Figure S4).
(B) The degree of mitochondrial fragmentation in the muscle cells of singled L1s arrested for 18 days predicted their ability to develop when fed (left panel). A score of 0, 1, or 2 represents no, intermediate, or high fragmentation levels per cell, and was used to calculate a total fragmentation score per animal. Averages are shown (right panel). ∗∗p = (see Figures S1F–S1H). Error bars represent SDs.
(C) The level of KIN-19::RFP aggregates in singled L1s arrested 10 days predicted their ability to develop when fed. Scale bar, 10 μm. ∗∗∗p < (see also Figure S4G).
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © Terms and Conditions

5. Figure 3
Feeding Erased All Signs of Aging in Arrested L1s, Except for Protein Aggregates, which Persisted
(A) Movement speed was restored after feeding L1s arrested 18 days. ∗∗∗p < L1∗, some worms started to grow.
(B) ROS-indicator DHE signal in the heads of singled 15-day-arrested L1s, each examined before and after 2–3 days feeding. Quantification is shown on right panel. G, intestinal granules; these exhibited an ROS signal even in day-1-arrested L1s; the levels over time may increase but were not quantified.
(C) Mitochondrial morphology in individual 18-day-arrested L1s over time, before and after feeding (quantification in Figure S5B). M, mitochondria; n, nucleus. Scale bar, 10 μm. Images are projections of 5–10 frames.
(D) Viability of recovering larvae increased markedly after feeding. L1 populations arrested for 1 or 16 days were fed for 0, 24, or 48 hr, and their further development was blocked with the DNA synthesis inhibitor 5-Fluoro-2′-deoxyuridine (FUdR) in order to measure their survival at different larval stages. Upper panel: experimental timeline. Lower graphics: feeding increased the length of time long-arrested larvae can survive. Survival curves of 1-day L1s (left panel) and 16-day L1s (right panel) shifted to plates with food. FUdR was added at the time indicated (0 hr, 24 hr, and 48 hr after feeding). n > 90 animals per curve; survival repeated once with similar findings (supporting material in Figure S6; replicates in Table S1).
(E) The number of polyQ35-YFP aggregates in 16-day-arrested L1s remained the same after 2 days of feeding and increased after 3 days of feeding, at the L4 stage. Each day assays a different fraction of the same population. Blue bar, average. ∗∗∗p < (see also Figures S5D and S5E for KIN-19::RFP aggregates).
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © Terms and Conditions

6. Figure 4
The ER-UPR Gene ire-1 Is Required for Arrested L1s to Recover and Regain Youthful Cellular Phenotypes
(A) The ER chaperone hsp-4/BIP was identified in a screen for RNAi clones that impaired the growth of long-arrested L1s, but not 1-day-arrested L1s, after feeding (see also Figures S7A and S7B and Table S3). RNAi control, vector-only (L4440) bacteria. ∗∗∗p < is the interaction p value for the two-way ANOVA test.
(B) Effect of the three major ER-UPR pathways on the recovery of L1s arrested for 10 days. Average of three experiments (n > 100 in each). ∗∗∗p < Error bars represent SDs.
(C) Compared to wild-type, L1-arrested ire-1(ok799) mutants soon lost the ability to develop when fed, independently of their survival. n > 100 for each time point (Table S2).
(D) The movement speed of ire-1(ok799) arrested L1s did not increase after feeding. Thrashing speed was measured in single 15-day-arrested L1s (0 hr) and after 12 and 24 hr feeding.
(E) Representative mitochondrial morphology of an ire-1(ok799) L1 photographed after 14 days of arrest and again after 4 days on food. Scale bar, 10 μm. n, nucleus; M, mitochondria.
(F) Longitudinal quantification of mitochondrial fragmentation shown in (E). No reversal was observed.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © Terms and Conditions

7. Figure 5
IRE-1’s Ability to Control L1 Recovery Partially Requires XBP-1
(A and B) The stress resistance of long-arrested L1s increased after feeding, prior to growth, particularly resistance to the ER stressor DTT. In contrast, the stress resistance of L1s starved for a short period of time, which is known to be increased relative to continuously fed L1s, was reduced after feeding (Jobson et al., 2015).
(A) DTT resistance (2.5 hr at 5 mM) of L1s arrested for 1 day and 16 days and after 12 hr feeding. n > 100, three times per condition, ∗∗p < 0.001, ∗∗∗p < After 12 hr feeding, day-1 L1s initiated growth; day-16 L1s did not.
(B) Heat-shock resistance (6 hr at 35°C) of L1s arrested for 1 and 16 days and then fed for 12 hr. ∗p = 0.029, ∗∗∗p < Twenty-four hours after feeding, heat-shock resistance increased slightly for both 1-day L1s and 16-day L1s (data not shown).
(C) Phsp-4::gfp fluorescence level in L1s arrested 1 day, 10 days, and 10 days and then fed 6 hr. The basal level of expression of Phsp-4::gfp decreases during the arrest (as with aging adults; data not shown) and goes back up during the recovery of long-term arrested L1s (in the intestine only). Arrows, auto-fluorescence of intestinal granules.
(D) An xbp-1 null mutation had a significant but relatively mild effect on the recovery of L1s arrested 10 days compared to that of ire-1(−) mutants. n > 100 in each of three experiments (average shown here). ∗∗∗p < , ∗p = (These xbp-1(−) animals were examined in parallel with the strains shown in Figure 4B; the same ire-1 data are shown again here for clarity.)
(E) L1 arrested mutants carrying ire-1(zc14), a partial loss-of-function mutation that strongly reduces hsp-4 induction in response to tunicamycin (Calfon et al., 2002), are only partially defective in recovery. n > 100 for each time point (Table S2).
(F) Treatment of wild-type day-10 L1s with 150 μM of the IRE-1 RNase inhibitor 4μ8C during the arrest and after feeding did not affect recovery. Development (body size) was assessed after 4 days of feeding. n = 50 in each of three trials.
(G) RT-PCR showing the splicing of xbp-1 mRNA after tunicamycin treatment (TM; 2 μg/mL). The splicing was inhibited in the presence of 150 μM 4μ8C inhibitor. Two independent samples are shown for each condition. PCR control, unc-15 primers.
(H and I) (H) Phsp-4::gfp expression levels measured by microscopy in the head of day-1 L1s increased after treatment with 2 μg/mL tunicamycin. The response was inhibited by 150 μM 4μ8C, as was (I) the ability of worms to develop to adulthood. ∗∗∗p < HS, heat shock.
Error bars represent SDs in (A), (B), (D), (F), (H), and (I).
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © Terms and Conditions

8. Figure 6
The Jun-N-Terminal Kinase KGB-1 Regulates L1 Recovery after Prolonged Arrest and Is Controlled by ire-1
(A) kgb-1(um3) null mutants had an impaired ability to recover from L1 arrest (less than half of wild-type; see Table S2). Like ire-1 mutants, their survival time was comparable to wild-type. n > 100 for each time point.
(B) KGB-1 phosphorylation was decreased in ire-1(ok799) mutants following a 10-day arrest. Protein extracts of L1s from wild-type and ire-1(ok799) mutants were prepared following either a brief or long L1 arrest before and after feeding. Pictures show a representative western blot of the same membrane probed with antibodies targeting KGB-1, phospho-KGB-1, or actin.
(C) kgb-1 is required for previously long-arrested L1s, but not briefly arrested L1s, to subsequently have normal adult lifespans. Lifespan of wild-type and kgb-1(um3) adults previously L1 arrested for 1 and 23 days or 1 and 16 days, respectively. n(blue) = 48 and n(red) = 62; p = n(orange) = 67 and n(green) = 99; p < (see Table S1).
(D) Muscle cells of kgb-1(um3) pre-adults (L4s) previously arrested 15 days as L1s had higher levels of mitochondrial fragmentation relative to those of kgb-1(um3) L4s arrested 1 day.
(E) Representative images of L4 worms from L1s arrested 1 day or 15 days showing fragmented mitochondria. Scale bar, 10 μm. n, nucleus; M, mitochondria. Scores “A–D” refer to the degree of mitochondrial fragmentation (see also Figures S1C and S1D).
(F) Each cell was attributed a mitochondrial fragmentation score of A to D (like in D; described in Figures S1C and S1D), and a score of 0, 1, 2, or 3 was respectively associated to it to calculate a fragmentation score per animal (details are in Experimental Procedures). Averages are shown. ∗∗p < Error bars represent SDs.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © Terms and Conditions