Supporting InformationKirienko et al. 10.1073/pnas.1424954112SI Materials and MethodsC. elegans Strains.C. elegans strains used in this study included N2Bristol (wild type), AU3 [nsy-1(ag3)], AY101 {acIs101[pDB09.1(pF35E12.5::GFP); pRF4(rol-6(su1006)]}, BR2430 [pdr-1(lg103)],ERT061 [zip-2(tm4248)], BR4006 [pink-1(tm1779); byEx655(Ppink-1::myc::pink-1::gfp; Pmyo-2::mCherry)], KU4 [sek-1(km4)],KU25 [pmk-1(km25)], [glp-4(bn2ts); zip-2(tm4248)], [glp-4(bn2ts);hif-1(ia4)], QU22 {bec-1(ok691); izEx5[pAR39.1(Pbec-1::bec-1::mRFP); pRF4(rol-6(su1006)]}, RB2547 [pink-1(ok3538)], SJ4103[zcIs14(myo-3p::GFPmt)], SS104 [glp-4(bn2ts)], VC942 [mboa-7(gk399)], VK1241 [vkEx1241(Pnhx-2::mCherry::lgg-1; Pmyo-2::GFP)], WY753 {fdEx142 [pJY323 (Phsp-16.1::GFP); pRF4(rol-6(su1006)]}, WY756 {fdEx139 [pJY312 (Phsp-16.1(dd)::GFP); pRF4(rol-6(su1006)]}, and ZD101 [tir-1(qd4)].Media conditions include NGM (1); modified NGM used for

plate-based infection with P. aeruginosa (SK) (2); modified liquidNGM used for liquid killing (LK) (3); brain–heart infusion media(Difco); tryptic soy agar media (Difco).Before experiments, worms were synchronized by hypochlorite

isolation of eggs from gravid adults, followed by hatching of eggs inS-basal. L1 larvae were transferred to NGM plates seeded withOP50 or NGM plates supplemented with 25 μg/mL carbenicillinand 1 mM IPTG that were seeded with appropriate RNAi strains.After transfer, worms were grown overnight at 15 °C, and thenshifted to 25 °C for 44–48 h before use. For strains that did nothave a sterile background, worms were reared on cdc-25.1(RNAi)before use in assays to guarantee sterility. Young adult worms wereused for all assays.All RNAi clones used in this study were from the Ahringer RNAi

library (4) and sequenced before use. RNAi treatment was per-formed by seeding 4,500 synchronized L1 larvae onto 10-cm NGMplates supplemented with carbenicillin and 1 mM IPTG. Wormswere used when they reached the young adult stage.

Imaging. For mitochondrial dynamics in C. elegans: synchronizedyoung adult SJ4103 worms (5) were to exposed to E. coli OP50 onNGM agar plates or in liquid kill medium (3) for 12 h, infected withP. aeruginosa PA14 on slow kill medium (2) for 12 h, exposed to P.aeruginosa PA14 in liquid kill medium for 12 h, exposed to vehicle(DMSO), 1 mM 1,10-phenanthroline, 1 mM ciclopirox olamine(Sigma) in S basal for 8 h, or exposed to partially purified pyoverdinin modified M9 medium (3) for 12 h. Worms were paralyzedwith 20 mM levamisole (Sigma) and mounted onto agar pads.Images were acquired and fluorescence was quantified usinga Zeiss AXIO Imager ZI microscope with a Zeiss AxioCam HRmcamera and AxioVision 4.6 (Zeiss) software. For quantification ofmorphology, at least 50 worms were used per condition perbiological replicate.For visualization in autophagy mutants, synchronized L1

SJ4103 larvae were dropped onto NGM agar supplemented withcarbenicillin and 1 mM IPTG that was seeded with E. coli con-taining RNAi constructs for bec-1/BECN, lgg-1/LC3, mboa-7/MBOAT7, or pink-1/PINK1. Young adults were washed off ofplates and exposed to 1 mM Phe in S basal or to vehiclealone (DMSO).For visualization of the BEC-1/BECN::RFP (6), mCherry::

LGG-1/LC3 (7), and PINK-1/PINK1::GFP (8) translational fu-sions, well-fed young adult worms were transferred from platesseeded with OP50 to freshly-made NGM plates supplementedwith 1 mM Phe for 8 h. Worms were paralyzed, mounted, andvisualized as above.

For visualization of the pF35E12.5::GFP (9) reporter, synchro-nized young adult AY101 worms were washed from NGM platesinoculated with E. coliOP50 and transferred to P. aeruginosa PA14on SK media plates, P. aeruginosa PA14 in LK media, or E. coliOP50 in LK media supplemented with RPW24 (10).For mitochondrial visualization in HEK293T cells, cells were

seeded at half-density on precleaned coverslips. When cellsreached ∼70% confluency, they were washed once with PBS andresuspended in serum-free DMEM supplemented with DMSO,0.5 mM Phe, or 0.1 mM ciclopirox olamine (Sigma). Cells weretreated for 12–16 h, and then directly stained with 200 nM MTR.Z-stacks were collected on an Olympus FluoView 1000 confocalmicroscope using 562-nm excitation. The same focal plane isshown for each cell on the basis of nuclear morphology in thebright-field channel. Unstained cells were used as controls.At least three biological replicates were performed for each

experiment. P values were determined by Student’s t test.

qPCR. Total DNA was extracted from synchronized worms washedfrom NGM plates seeded with E. coli OP50 and treated with 1 mMphenanthroline or DMSO in S basal. Primers were designed for thend1 mitochondrial gene and act-3, as previously described (11).DNA was used for quantitative PCR using SYBR Green (BioRad),and mitochondrial gene level was normalized to nuclear genenumber using the ΔΔCt method (12). Thermocycler parameterswere as for qRT-PCR.Before DNA extraction, mammalian cells were washed with PBS

and media was replaced with serum-free DMEM that was supple-mented with 0.25% (vol/vol) DMSO, 0.5 mM Phe, or 0.1 mMciclopirox olamine for 12 h. Primers were designed for human nd1 andnd4 genes, and mitochondrial genes were normalized to tubulin usingthe ΔΔCt method.For each experiment at least three biological replicates were per-

formed. P values were calculated by Student’s t test. Primer se-quences are available upon request.

qRT-PCR. RNA purification and qRT-PCR were performed as pre-viously described (13, 14). Human ESRE genes were identified usinga reciprocal BLAST search as previously described (14). BeforeRNA extraction, cells were treated with Phe, ciclopirox olamine, orDMSO as described for qPCR.Primer sequences are available upon request. For each ex-

periment at least three biological replicates were used. P valueswere derived from Student’s t test.

Immunoblotting. HEK293T cells were treated with iron chelators orvehicle (DMSO), as described for qPCR. After treatment, cells wereharvested by physical disruption and lysed with RIPA buffer. Sampleswere analyzed by SDS/PAGEon 4–20% gradient TGX gels (BioRad)before transfer to PVDF membranes (Millipore). Membranes wereblocked with 5% (wt/vol) nonfat dry milk in Tris-buffered saline.Primary antibodies included anti–α-tubulin at 1:1,000 (SAB3501071;Sigma), anti-NRF1 at 1:1,000 (ab175932; Abcam), and anti–Phos-pho-NRF-2 (ab76026; Abcam) at 1:5,000. Secondary antibody wasanti-rabbit (A0545; Sigma). Detection was performed via chem-iluminescence (SuperSignal West Pico; Pierce Thermo Scientific) onCarestream BioMax MR film.For each experiment, at least three biological replicates

were used.

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1. Stiernagle T (2006) Maintenance of C. elegans. WormBook 11:1–11.2. Tan MW, Mahajan-Miklos S, Ausubel FM (1999) Killing of Caenorhabditis elegans by

Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. ProcNatl Acad Sci USA 96(2):715–720.

3. Kirienko NV, Cezairliyan BO, Ausubel FM, Powell JR (2014) Pseudomonas aeruginosaPA14 pathogenesis in Caenorhabditis elegans. Methods Mol Biol 1149:653–669.

4. Kamath RS, et al. (2003) Systematic functional analysis of the Caenorhabditis elegansgenome using RNAi. Nature 421(6920):231–237.

5. Benedetti C, Haynes CM, Yang Y, Harding HP, Ron D (2006) Ubiquitin-like protein 5positively regulates chaperone gene expression in the mitochondrial unfolded pro-tein response. Genetics 174(1):229–239.

6. Rowland AM, Richmond JE, Olsen JG, Hall DH, Bamber BA (2006) Presynaptic termi-nals independently regulate synaptic clustering and autophagy of GABAA receptorsin Caenorhabditis elegans. J Neurosci 26(6):1711–1720.

7. Miedel MT, et al. (2012) A pro-cathepsin L mutant is a luminal substrate for endo-plasmic-reticulum-associated degradation in C. elegans. PLoS ONE 7(7):e40145.

8. Sämann J, et al. (2009) Caenorhabditits elegans LRK-1 and PINK-1 act antagonisticallyin stress response and neurite outgrowth. J Biol Chem 284(24):16482–16491.

9. Bolz DD, Tenor JL, Aballay A (2010) A conserved PMK-1/p38 MAPK is required inCaenorhabditis elegans tissue-specific immune response to Yersinia pestis infection.J Biol Chem 285(14):10832–10840.

10. Pukkila-Worley R, et al. (2012) Stimulation of host immune defenses bya small molecule protects C. elegans from bacterial infection. PLoS Genet 8(6):e1002733.

11. Bratic I, et al. (2009) Mitochondrial DNA level, but not active replicase, is essential forCaenorhabditis elegans development. Nucleic Acids Res 37(6):1817–1828.

12. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25(4):402–408.

13. Kirienko NV, Fay DS (2007) Transcriptome profiling of the C. elegans Rb orthologreveals diverse developmental roles. Dev Biol 305(2):674–684.

14. Kirienko NV, Fay DS (2010) SLR-2 and JMJC-1 regulate an evolutionarily conservedstress-response network. EMBO J 29(4):727–739.

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Fig. S2. Iron chelators reduce mitochondrial mass. Flow vermimetry (with a BIOSORT COPAS) of worms exposed to Phe (1 mM), CPX (1 mM), or DMSO andlabeled with nonyl acridine orange (10 μM). Fluorescence was normalized to worm size, as based on time of flight. Three biological replicates were performed,n = 5,000 for each biological replicate. Error bars represent SEM.

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