C. elegans Strains
Nematode strains used were described previously 
or received from the Caenorhabditis elegans
Genetics Center CGC (St Paul, MN). All strains were maintained following standard methods on OP50 bacteria plates. Strains used in this study include: tdp-1(ok803) and tdp-1(ok781)
both outcrossed 5 times to N2 prior to use, daf-2(e1368)
, daf- 2(e1370)
, gpIs1[hsp-16.2::GFP], oxIs12[unc-47p::GFP;lin-15(+)], xqIs93[tdp-1p::TDP-1::GFP], xqIs132[unc-47p::TDP-43-WT;unc-119(+)], xqIs133[unc-47p::TDP-43[A315T];unc-119(+)], xqIs173[unc-47p::FUS-WT;unc-119(+)], xqIs98[unc-47p::FUS[S57Δ];unc-119(+)], zIs356[daf-16p::daf-16-gfp; rol-6], zcIs4[hsp-4p::GFP], zcIs9[hsp-60p::GFP],gpIs1[hsp-16.2p::GFP] and zcIs13[hsp-6p::GFP].
Sixty synchronized L4 were grown on OP50 bacteria plates (20 animals/plate) and three independent assays were performed. Lifespan analyses were performed at 20°C and 25°C and worms were scored every 1–2 days from adult day 1 until death. Worms were scored dead if they didn't respond to tactile or heat stimulus.
RNAi-treated strains were fed with E. coli (HT115) containing an Empty Vector (EV), daf-16 (R13H8.1), or tdp-1 (F44G4.4) RNAi clones from the ORFeome RNAi library (Open Biosystems). RNAi experiments were performed at 20°C. Worms were grown on NGM enriched with 1 mM Isopropyl-b-D-thiogalactopyranoside (IPTG). For lifespan, worms were transferred to RNAi 5-fluorodexyuridine (FUDR, 12.5 mg/L) plates at adult day 1 until death. Worms were declared dead if they didn't respond to tactile or heat stimulus. Experiments were conducted with 20 animals/plate by triplicates.
Dauer Formation Assay
Young adult daf-2(e1370) were allowed to lay eggs overnight at 20°C. The eggs were then transferred to 25°C and scored for dauer formation 5 days later. Three different trials on different days were performed.
Stress tests were performed at 20°C (oxidative, osmotic and UV stress), 25°C (hypoxia) and 37°C (thermal stress). Worms were grown on NGM and transferred to NGM plates + 240 µM juglone (oxidative stress), or NGM plates + 10 mM Hydrogen peroxide (oxidative stress), or NGM plates + 400 mM NaCl (osmotic stress), or NGM plates + 611 mM Sorbitol (osmotic stress), all at adult day 1. For the oxidative, osmotic and thermal stress assays, worms were evaluated for survival every 30 minutes for the first 2 hours and every 2 hours after up to 14 hours; for sorbitol we also performed a test over 48 hours, starting the counts after 14 hours on the compound. For UV irradiation, adult day 1 worms were transferred to NGM plates without any food source and exposed to UV (1200 J/m2). Worms were then transferred to NGM plates with OP50 bacteria and died animals were counted every 2 hours till 14 hours after irradiation. For hypoxia experiments young adult animals were transferred to a new plate and subjected to low oxygen conditions with the AnaeroPack system (Mitsubishi Gas Chemical America) for 24 hours and assayed for survival. For all experiments nematodes were scored as dead if they were unable to move in response to heat or tactile stimuli. For all tests worms, 20 animals/plate by triplicates were scored.
Gateway system (Invitrogen) compatible tdp-1 promoter and open reading frame plasmid clones were obtained from Open Biosystems and recombined with plasmid pDES-MB14 (kindly donated by M. Vidal, Harvard), and verified by sequencing to create a tdp-1p::TDP-1::GFP plasmid, which was injected at 5 ng/µl into unc-119(ed3) animals along with myo-3p::mCherry, myo-2p::mCherry comarkers at 5 ng/µl and wild type transformants expressing GFP were kept. The transgene was integrated using UV radiation and wild type, GFP positive animals were kept for further study. Multiple stable transgenics were isolated and outcrossed to N2 4 times before use. Strain XQ93 xqIs93[tdp-1p::TDP-1::GFP] was used in this study.
For visualization of TDP-1::GFP
animals, M9 buffer with 5 mM levamisole was used for immobilization. Animals were mounted on slides with 2% agarose pads. TDP-1::GFP
expression was visualized with a Leica CTR 6000 and a Leica DFC 480 camera. L4 animals were grown on NGM plates and transferred to NGM plates + 240 µM juglone (oxidative stress) or NGM plates + 400 mM NaCl (osmotic stress) for 90 minutes, and examined for fluorescence with the Leica system described above. Some animals were also stained with DAPI (1
1000, diluted in 1× PBS). Image processing was done with Adobe Photoshop. For images of TDP-1::GFP alone, images were converted to black and white and the images reversed to allow for better contrast and visualization. Quantification of TDP-1::GFP levels was done with ImageJ (NIH) and the mean and SD was calculated from 5 images for each strain and experimental condition. For visualization of DAF-16::GFP
, hsp-4p::GFP, hsp-6p::GFP, hsp-16.2p::GFP and hsp-60p::GFP
animals, M9 buffer with 5 mM levamisole was used for immobilization. Animals were mounted on slides with 2% agarose pads and examined for fluorescence with the Leica system described above.
Dihydrofluorescein Diacetate Assay
For visualization of oxidative damage in the transgenic strains the worms were incubated on a slide for 30 minutes with 5 mM dihydrofluorescein diacetate dye (Sigma-Aldrich) and then washed with 1× PBS three times. After the slide was fixed fluorescence was observed with the Leica system described above.
RNA was extracted with an RNAeasy kit (Qiagen) and reverse transcribed with QuantiTect (Qiagen). Primers used include: ctl-1 forward, AGGTCACCCATGACATCACCAAGT; ctl-1 reverse, GAT TGCGCTTCAGGGCATGAATGA; ctl-2 forward, TTCGCTGAGTTGAACAATCCG; ctl-2 reverse, GTTGCTGATTGTCATAAGCCATTGC; tdp-1 forward, AAAGTGGGATCGAGTGACGAC; tdp-1 reverse, GACAGCGTAACGAATGCAAAGC; sod-3 forward, CGAGCTCGAACCTGTAATCAGCCATG; sod-3 reverse, GGGGTACCGCTGATATTCTTCCACTTG; act-3 forward, GTTGCCGCTCTTGTTGTAGAC; act-3 reverse, GGAGAGGACAGCTTGGATGG.
Worms were collected in M9 buffer, washed 3 times with M9 and pellets were placed at −80°C overnight. Pellets were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris pH 7.4, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate) + 0.1% protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A, 10 mg/ml chymostatin LPC;1/1000). Pellets were passed through a 271/2 G syringe 10 times, sonicated and centrifuged at 16000 g. Supernatants were collected.
For TDP-43 and FUS transgenics, soluble/insoluble fractions, worms were lysed in Extraction Buffer (1 M Tris-HCl pH 8, 0.5 M EDTA, 1 M NaCl, 10% NP40 + protease inhibitors (LPC;1/1000). Pellets were passed through a 271/2 G syringe 10 times, sonicated and centrifuged at 100000 g for 5 minutes. The soluble supernatant was saved and the remaining pellet was resuspended in extraction buffer, sonicated and centrifuged at 100000 g for 5 minutes. The remaining pellet was resuspended into 100 µl of RIPA buffer, sonicated until the pellet was resuspended in solution and saved.
All supernatants were quantified with the BCA Protein Assay Kit (Thermo Scientific) following the manufacturer instructions.
Worm RIPA samples (175 µg/well for transgenic worms; 15 µg/well for non transgenics) were resuspended directly in 1× Laemmli sample buffer, migrated in 10% polyacrylamide gels, transferred to nitrocellulose membranes (BioRad) and immunoblotted. Antibodies used: rabbit anti-TDP-43 (1
200, Proteintech), rabbit anti-FUS/TLS (1
200, Abcam), rabbit anti-TDP-1 (1
500, Petrucelli laboratory) and mouse anti-Actin (1
10000, MP Biomedical). Blots were visualized with peroxidase-conjugated secondary antibodies and ECL Western Blotting Substrate (Thermo Scientific). Densitometry was performed with Photoshop (Adobe).
For lifespan and stress-resistance tests, survival curves were generated and compared using the Log-rank (Mantel-Cox) test, and 20–30 animals were tested per genotype and repeated at least three times. For progeny counts, dauer-formation assays and hypoxia tests the mean and SEM were calculated for each trial and two-tailed t-tests were used for statistical analysis.