“Slow killing” P. aeruginosa infection assays were performed as previously described (Tan et al. 1999 (link)). A single colony of P. aeruginosaPA14 was inoculated into 3 ml of Luria-Bertani (LB) medium, and allowed to incubate at 37° for 14–15 hr; 10 μl of this culture was added to 35-mm tissue culture plates containing 4 ml of slow kill agar (0.35% peptone, 0.3% sodium chloride, 1.7% agar, 5 μg/ml cholesterol, 25 mM potassium phosphate, 1 mM magnesium sulfate, 1 mM calcium chloride). Plates were incubated for 24 hr at 37°, and 24 hr at 25°. At 1–2 hr before the start of the assay, 0.1 mg/ml 5-fluorodeoxyuridine (FUDR) was added to the medium to prevent progeny from hatching. A total of 40–50 animals at the young L4 larval stage were picked to each of three or four assay plates per experimental condition. C. elegans was prepared for the pathogenesis assays in the manner described above for the nanoString experiment to ensure that stage-matched nematodes were used for these experiments. Animals were scored as live or dead on a daily basis by gently touching them with a platinum wire. Worms that crawled onto the wall of the tissue culture plate were eliminated from the analysis. P. aeruginosa killing assays were conducted at 25°.
The development assay presented in Figure 1 was conducted by placing approximately 100 hypochlorite-synchronized L1 larval stage animals of the indicated genotype on “slow kill” medium plates (Tan et al. 1999 (link)) containing 140 μM R24 or the solvent control DMSO (1%), and monitoring development to the young adult stage for 3 d at 20° on two replicate plates per condition. For the development assays conducted with RNAi bacteria (Figure 4B), two L4 larval stage animals of the indicated genotype were allowed to lay their brood on RNAi bacteria at 15°. Plates were then transferred to 20° for 3 d. The stage of approximately 300 animals on each of three replicate plates per condition was recorded, and the percentage of animals at the L4 larval stage was reported. Representative animals from each condition in this experiment were photographed. For the experiments with the xbp-1(zc12) mutant (Figure S4), four animals were allowed to lay their brood for 8 hr at 20° in the presence or absence of 70 μM R24. C. elegans carrying the zcIs4 transgene was used as the control for these experiments.
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