5 fluoro 2 deoxyuridine fudr

The transgenic strains, GRU101[myo-2p::yfp] and GRU102[myo-2p::yfp + unc-119p::Aβ1–42], previously reported in Fong et al. (2016) (link) were used in all experiments. These animals were raised at 20°C throughout the experiment and transferred to nematode growth medium (NGM) plate supplemented with 250 uM 5-fluoro-2′-deoxyuridine (FUDR; Sigma-Aldrich, St. Louis, USA) to prevent progeny production. Age-synchronized animals were obtained for all experiments via hypochlorite bleaching. Young animals refer to day four post-bleaching while old animals refer to day 12 post-bleaching. For Metformin treatment, Metformin Hydrochloride (Sigma-Aldrich, St Louis, United States) was dissolved in water and added into NGM agar to a final concentration of 50 mM. Animals were transferred to Metformin-containing plate on day three post-hatching at L4 stage. For RNAi treatment, RNAi feeding clones were obtained from the C. elegans ORF-RNAi Library (Vidal) from Source BioScience. L4 animals were fed with HT115(DE3) bacteria expressing either the empty vector (EV) or RNAi clone T22B11.5 (for aKGDH knockdown), on NGM plates containing 2 mM IPTG and 100 ug/ml Carbenicillin.

The C. elegans lifespan assay was performed by transferring 15 young adult (L4 stage) worms per bacterial species to three mNGM/FUdR plates. 5-Fluoro-2′-deoxyuridine (FUdR, Sigma Aldrich, St. Louis, MO, USA) (50 μM) was added to the plates41 (link), and E. coli OP50 and P. freudenreichii were seeded on them. The plates were incubated at 25 °C, and the numbers of live or dead worms were scored every 24 h. Worms were considered “dead” when they failed to respond to a gentle touch with a worm picker. Nematodes that crawled off the plates and showed non-natural deaths, such as bagging or adhering to the wall of the plate, were censored42 (link). For the first 3 days, worms were transferred to fresh mNGM/FUdR every day, and then they were transferred once per week for the rest of the experiment to maintain a sufficient food source. Experiments were performed at least in triplicate, and more than 100 worms for each bacterial species were used in the longevity assay.
The mean lifespan was estimated using the following formula43 (link):

where j is the age category (day), dj is the number of worms that died in the age interval (xj, xj1), and n is the total number of worms. The standard error (SE) of the MLS estimate was calculated using the following equation:

The toxic effect of different concentrations of GNC was evaluated. Age-synchronized day 1 adult N2 C. elegans were incubated with a series of GNC concentrations, from 0.00394 mg/mL to 78.8 mg/mL, in S-complete liquid medium (a liquid culture medium for C. elegans) [23 ]. A final concentration of 400 μM 5-fluoro-2′-deoxyuridine (FUDR, Sigma-Aldrich Co., St. Louis, MO, USA) was added to the medium to block progeny development. Survival was assessed after 48 hours of treatment with GNC. The nematodes were considered dead when they failed to respond to touch using a platinum loop. Ninety worms at each concentration were tested, and the experiment was performed three times independently.

Lifespan assays were performed as described previously [12 (link)]. Briefly, wild-type (N2) and fat-6-overexpressing worms (IJ508 yhEx112 [ges-1p::fat-6::GFP, odr-1::RFP]) were maintained on Escherichia coli (OP50)-seeded nematode growth medium (NGM) agar plates at 20°C. Synchronized wild-type and fat-6-overexpressing animals were transferred onto OP50-seeded NGM agar plates containing 10 μM 5-fluoro-2′-deoxyuridine (FUdR, Sigma, St Louis, MO, USA) at day 1 adult stage to prevent progeny from hatching. Worms that did not respond to gentle touching by a platinum wire were counted as dead. Animals that crawled off the plates, ruptured, or burrowed were censored but included in the statistical analysis. The movement-capacity data were adopted from our previously published paper [9 (link)].

C. elegans Bristol-N2 was maintained at 20°C until adult stage on nematode growth medium (NGM) plates. To collect eggs, fertilized C. elegans adults were incubated with alkaline lysis buffer while being shaken for 20 min at room temperature and subsequently washed twice with M9 buffer. To arrest the larvae to L1 stage, the C. elegans eggs were transferred for hatching on new NGM plates and incubated at 20°C for 20 h without food. L1 synchronized worms were transferred to fresh NGM with nonpathogenic E. coli OP50 for 48 h to mature up to the L4 stage. Five worms per well were then distributed on 24-well plates with NGM agar with 40 μM 5-fluoro-2′-deoxyuridine (FUDR) (Sigma) to prevent progeny production (55 (link)). Additionally, 500 μl HBSS medium (Lonza) was added to each well. Sets of five wells either contained 2 × 10⁸E. coli OP50 cells/well or 3 × 10⁵C. albicans SC5314 cells/well. Plates were incubated at 25°C in normoxic and hypoxic (1% O₂) cell incubators with 5% CO₂ for 48 h, and C. elegans survival was subsequently recorded with a Nikon SMZ1500 stereomicroscope.