7918 Nucleic Acids Research, 2012, Vol. 40, No. 16 RNA interference RNAi-mediated interference was carried out by feeding. HT115 (DE3) RNase III-deﬁcient Escherichia coli engin- eered to express dsRNA for eat-3 (D2013.5), drp-1 (T12E12.4), pink-1 (EEED8.9) and pdr-1 (K08E3.7) were obtained from Geneservice (Cambridge, UK) or Open Biosystems (Huntsville, AL, USA). atg-18 (F41E6.13), bec-1 (T19E7.3), fzo-1 (ZK1248.14) and unc-51 (F41E6.13) were retrieved from the Vidal C. elegans RNAi library (58), and pL4440 (empty vector, negative RNAi control) and unc-22 (ZK617.1, positive RNAi control) were retrieved from the Ahringer C. elegans RNAi library (59). All RNAi vectors were sequenced to verify the identity of the insert. RNAi stocks were selected on LB plates containing 15 mg/ml tetracycline and 100 mg/ml ampicillin (Sigma Aldrich). Bacteria were grown in LB containing 50 mg/ml ampicillin (ﬁnal concentration) for 8 h and were added to NGM plates containing 50 mg/ml ampicillin and 1 mM isopropyl b-D-1-thiogalactopyranoside (Sigma Aldrich). With the exception of eat-3, fzo-1 and drp-1 RNAi knockdown, L1 glp-1 nematodes were synchronized as described earlier in the text and grown on RNAi-seeded NGM plates for two generations. Nematodes were grown at 15 C throughout the experiment except for a 15-h period between the L3 and L4 stage during which nematodes were transferred to 25 C for sterilization (60). At the adult stage of the second generation, glp-1 nematodes were dosed with 50 J/m2 UVC and lysed imme- diately and 120 h after the UVC dose for DNA damage analysis. For eat-3, fzo-1 and drp-1 RNAi knockdown, an identical procedure was carried out except over one generation instead of two because of embryonic lethality and larval arrest in F1 progeny of nematodes treated with eat-3, fzo-1 (61) and drp-1 (62) RNAi. Mitochondrial morphology and autophagy analysis Synchronized young adult N2 myo-3::matrixGFP and N2 LGG-1::GFP nematodes were maintained and UVC exposed as described earlier in the text except at 20 C instead of 25 C. At recovery periods, 6–10 nematodes were picked onto 10% agar pads with 10 ml of 10 mM levamisole (Sigma Aldrich, dissolved in water). Single- plane images were taken of muscle cell mitochondria and seam cells from ﬁve to 10 nematodes per experiment in two, time-separated experiments on a Zeiss 780 confocal microscope at 63 magniﬁcation. Approxi- mately 9–15 seam cells and 25–50 muscle cells were analyzed per treatment per time point. LGG-1::GFP foci in each seam cell were counted manually. An overall effect of treatment determined by ANOVA (P < 0.0001) (Statview 5.0.1) and a difference between speciﬁc treat- ments was determined by Fisher’s PLSD. Images of muscle cell mitochondria were analyzed using MetaMorph Premier and methods described in (63,64). Brieﬂy, back- ground subtraction, a top hat morphological ﬁlter and a threshold were applied to images. After converting to a binary image, mitochondrial area excluding the area of ‘holes’, perimeter and form factor for each muscle cell was measured using Integrated Morphology Analysis. Larval arrest protocol L1 worms were plated in equal amounts on unseeded, non-peptone (to prevent growth of bacterial contamin- ation), K-agar plates with or without ethidium bromide (Sigma Aldrich) for 48 h. L1 nematodes were starved during this period to prevent development. L1 worms were exposed to UVC at 0, 24 and 48 h after plating. Immediately following the 48-h UVC dose, worms were transferred to 24-well, seeded, K-agar plates (1–3 worms/ well, 12 wells/treatment, 1 plate/strain) with or without ethidium bromide. For 3-methyladenine (3-MA) exposure following the 48 h UVC dose, worms were transferred to 24-well, seeded, liquid K+ as described earlier in the text with or without 3-MA (Sigma Aldrich, dissolved in dH2O). Every 24 h for 96 h, each worm was staged and the percentage of nematodes that reached L4 was determined based on the presence of the vulval crescent. All mutant strains and/or treatments were screened in a minimum of three time-separated experi- ments and the % L3 from each experiment was considered a biological replicate. % L3 across all time points was compared between strains (if applicable) and treatments by repeated measures ANOVA (Statview 5.0.1). Given a signiﬁcant interaction in the repeated measures ANOVA, ANOVA was used to determine overall effects and interactions at each time point followed by Fisher’s PLSD, if warranted. Relative ATP analysis PE255 nematodes were dosed according to the UVC dosing scheme described earlier in the text (Larval Arrest Protocol), and ATP analysis was conducted imme- diately after the third UVC dose (0 h) or 24 h after the third dose and placement on food. Each experiment had two biological replicates dosed on separate no-peptone, no-food plates and three time-separated experiments were performed (n = 6). Luminescence and GFP were measured using a FLUOstar Optima (BMG Labtech) on 100 nematodes in 100 ml of K-media added to a 96-well plate with two to four technical replicates/biological replicate. GFP was measured initially using 485/520 ﬁlter set. Following GFP measurement, 50 ml of lumines- cence buffer (citrate phosphate buffer pH 6.5, 0.1 mM D-luciferin, 1% DMSO and 0.05% Triton-X, ﬁnal concen- trations) was injected into each sample, and luminescence was measured 3 min after injection using a 590 nm emission ﬁlter. Luminescence was normalized to GFP ﬂuorescence intensity to account for differences in number of worms per well. Two-way ANOVA was used to determine if there was an effect of treatment and/or recovery on % control luminescence (Statview 5.0.1). After establishing that there was a treatment by recovery effect (P = 0.0017), the effect of treatment at each time point was assessed using Fisher’s PLSD. Oxygen consumption analysis N2 nematodes were treated with 10 J/m2 UVC as described earlier in the text (Larval Arrest Protocol) and oxygen consumption was measured at 0, 24 and 48 h post