The following strains were used: N2 Bristol wild type, JF97 mtl-1
), VC128 mtl-2
), JF27 cdr-1
), and VF2 pcs-1(tm1748)
. Each mutant strain was outcrossed three times to the N2 Bristol wild type strain. Mutants were selected using genotyping PCR (see below). All strains were maintained on nematode growth medium (NGM) agar plates containing OP50 Escherichia coli
at 20°C (Sulston and Hodgkin, 1988
), unless otherwise indicated.
Construction of double and triple mutants.
To create mtl-1
, and cdr-1
double and triple mutants, recombination events were selected from crosses involving mtl
), and cdr-1
). To create the double mutants, mtl-1
males were crossed with mtl-2
hermaphrodites, and cdr-1
males were crossed with mtl-2
hermaphrodites. To create the triple mutant, mtl-2
hermaphrodites were crossed with mtl-1
males, and mtl-1
hermaphrodites were crossed with cdr-1
males. From each cross, one F2 L4 larva was placed on an NGM agar plate and 12–20 F2 lines were isolated for each cross. Genomic DNA was isolated from F3 progeny using single worm lysis buffer (50mM KCl, 10mM Tris-HCl, pH 8.3, 2.5mM MgCl2
, 0.45% NP-40, 0.45% Tween 20, and 0.05 µg/µl proteinase K) and incubated for 1h at 65ºC followed by a 20-min incubation at 95ºC. PCR was performed to identify recombinants that were homozygous for the deletion mutations. Products generated using gene-specific primers are presented in Supplementary table 1
. Three independent PCR reactions were performed for each line to confirm the presence of the deletion. This PCR assessment was repeated throughout the experiments to ensure that all lines had the correct genotype.
Brood size analysis assay.
One L4 hermaphrodite was placed on an NGM agar plate. Adults were transferred every 24h for 3 days. Larva and dead embryos were counted on each plate 24h after removal of the adult. To test the effect of temperature on brood size, plates were placed at 20°C, 25°C, and 27°C for the duration of the experiment. For heat shock, L4 larvae were placed at 33°C for 2h, removed, and then placed at 20°C till the end of the experiment. For brood size analysis in response to cadmium, 100mM CdCl2
dissolved in K medium (32mM KCl and 51mM NaCl) (Williams and Dusenbery, 1990
) was added to NGM agar plates to a final plate concentration of 50, 75, and 100µM. Preliminary studies showed no noticeable difference between exposures on K agar plates versus NGM agar plates. The cadmium was allowed to dry for 24h, and the plates were then seeded with OP50 E. coli
. One L4 hermaphrodite was placed on a plate and allowed to grow at 20°C. Adults were transferred every 1.5 days due to the slower egg-laying rate in cadmium-exposed animals. Larva and dead embryos were counted on each plate 24h after removal of the adult. For all conditions, total brood size refers to the number of larvae counted over the length of the experiment. Percent embryonic lethality was calculated by dividing the number of dead embryos by the total brood size (dead embryos and larva) multiplied by 100. For all conditions, n
= 12 (three independent experiments).
Statistical analysis of brood size was accomplished by first determining whether brood size and embryonic lethality were normally distributed. Brood size was normally distributed and was therefore analyzed using parametric statistical methods. Analysis of variance (ANOVA) followed by Dunnett’s test at α = 0.05 was used to compare the single, double, and triple mutant strains with the wild type. Two-sample t-tests were used to compare brood sizes of single mutants with double mutants, single mutants with the triple mutant, and double mutants with the triple mutant.
Percent embryonic lethality was not normally distributed and could not be normalized with logarithm, square root, or arcsine transformations; therefore, nonparametric statistical methods were used. Kruskal-Wallis ANOVA was used to test the equality of percent embryonic lethality across all mutants, followed by Mann-Whitney tests to compare pairs of mutants, including comparisons of each mutant with the wild type, single mutants with double mutants, single mutants with the triple mutant, and double mutants with the triple mutant. Separate analyses were conducted for each temperature and cadmium concentration.
Assay for the bag phenotype. The bag phenotype is characterized by the buildup of embryos in a hermaphrodite due to its inability to release embryos. Twenty-five L4 hermaphrodites were placed on an NGM agar plate containing cadmium at various concentrations and incubated at 20°C. After 48h, adults were transferred to fresh plates, to be removed from their progeny, and subsequently transferred every 24h for three more days. During each transfer, any nematodes displaying the Bag phenotype were noted and removed from the experiment. On day 6, the number of living adults was determined. For each condition, five plates were tested.
Growth analysis assay.
The Complex Object Parametric Analyzer and Sorter (COPAS) Biosort was used to determine the effects of cadmium on growth as previously described (Boyd et al., 2010
). Mutant strains were synchronized using the alkaline hypochlorite preparation as previously described (Khanna et al., 1997
). Briefly, gravid adults were incubated in 1% Clorox containing 250µM NaOH. Embryos were collected and washed with K medium. They were then placed in K-plus medium (K medium plus 5 µg/ml cholesterol, 3mM CaCl2
, and 3mM MgSO4
) (Williams and Dusenbery, 1990
) and allowed to hatch overnight to synchronize at the L1 larval stage.
Using the COPAS Biosort, 50 L1 larvae were placed in each well of a 96-well microtiter plate. Each well contained K-plus medium, OP50 E. coli, and cadmium at the indicated concentrations. Nematodes were then incubated at 20°C for 48h. Visual inspection of the animals was made, and the COPAS Biosort was then used to determine the time of flight, which is a measure of the length of the nematode, and extinction, which is a measure of the optical density of the nematode. Both these measurements correspond to the developmental stage of the nematode. Under control conditions, wild type L1 larvae developed to the L4 stage in 48h. Three independent experiments (n ~ 200 L1/experiment) were conducted at all concentrations.
Statistical analysis of growth was accomplished as previously described (Boyd et al., 2010
). Dunn’s multiple testing procedure (Cardillo, 2006
) was used to compare mutant strains with the wild type at each cadmium concentration. Dunn’s test is nonparametric, not requiring normally distributed data, and sets an overall significance level of 0.05 for all comparisons to a given reference group. After comparing all mutant strains with wild type, each double mutant and the triple mutant were compared with their component single mutants. For example, the double mutant, mtl-2, cdr-1
, was compared with the single mutants mtl-2
Detection of PCs using liquid chromatography-mass spectrometry. Age-synchronized L1 nematodes were placed on NGM agar plates containing 0 or 12µM CdCl2 and incubated at 20°C for 48h. Nematodes were then collected by centrifugation and rinsed twice with K medium to remove all bacteria from the gut of the nematodes. Final nematode pellets (~400 µl) were transferred to 2.5-ml tubes, immediately frozen, and stored at −80°C until use.
To prepare extracts for liquid chromatography-mass spectrometry (LC/MS) analysis, frozen pellets were thawed at 4°C and 1ml of 0.6mM tris(2-carboxyethyl)phosphine hydrochloride in 100% methanol (Sigma-Aldrich, St Louis, MO) was added and mixtures were homogenized using a Mini-Bead Beater (Biospec Products, Bartlesville, OK). Homogenates were centrifuged and supernatants were then transferred to clean tubes containing 60 µl of freshly prepared 266mM N-ethylmaleimide (NEM) (Sigma-Aldrich). Supernatants and pellets were stored at −80°C until use.
Levels of GSH, PC2, and PC3 were determined in derivatized C. elegans supernatants using an Agilent Technologies 6224 TOF LC/MS equipped with an Ascentis 0.2 × 50mm 2.7µM C-18 column (Supelco, Sigma-Aldrich). Compounds were separated with a 0–90% linear, acetonitrile gradient over 26min. Data were analyzed using Agilent Mass Hunter Qualitative Analysis Software, Version B.03.01 (Agilent Technologies, Santa Clara, CA). To determine the exact masses and retention times of GSH-NEM, PC2-NEM, and PC3-NEM, NEM derivatives of GSH, PC2, and PC3 standards were prepared and analyzed as described earlier. Reduced and oxidized GSH treated with tris(2-carboxyethyl)phosphine hydrochloride or NEM gave identical chromatographic and spectral features. Extracted ion chromatograms for the exact masses of GSH-NEM, PC2-NEM, and PC3-NEM were integrated and compared with protein concentration (see below). Several independent experiments were analyzed by normalizing data for each experiment to PC2-NEM extracted ion chromatogram integrated peak area observed in wild type C. elegans.
Soluble protein was prepared by adding 0.9mM NaOH to the insoluble pellet and heating the mixture at 80°C for 1h with occasional shaking. The solution was then cooled and centrifuged, and the protein concentration was determined using the Bradford protein quantification assay following the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA). To estimate a dilution factor from median measured protein absorbance for each strain and repetition of the experiment, second-degree polynomials were fit to the protein concentration data (Olson and Markwell, 2007
). These dilutions were subsequently used as multiplicative scaling factors for median peak heights measured using LC/MS. For each strain and cadmium treatment, the base-10 logarithm of the product of the dilution factor and peak height was used for statistical analyses. These normalized data are referred to as the “scaled peak height.” To detect differences between median scaled peak heights for different strains and treatments for PC2 and PC3, Kruskal-Wallis rank sum tests were performed. Pairwise differences were then assessed using Mann-Whitney nonparametric two-sample tests. One-sided Mann-Whitney tests were used to assess cadmium treatment effects; two-sided tests were used to assess strain effects within untreated and within cadmium-treated nematodes.