(A) Screening strategy to isolate elo-5(−) suppressors. Green-colored C. elegans carry the extrachromosomal array containing copies of the elo-5(+), sur-5-gfp, and rol-6(dn) genes. (B) Percentages of animals reaching adulthood (bypass L1 arrest) when fed with/without C17ISO supplement. elo-5(−);ku540 mutant animals reach adulthood without C17ISO supplement. Error bar, SD. (C) Images showing that elo-5(−) animals arrested at L1 and elo-5(−);ku540 animals reached adulthood. (D) Mass spectrometry by precursor scan m/z = −241.2 showing no detectable C15FA-containing lipids in elo-5(−);ku540 animals. Numeric data for the levels of PA and PE are shown in Figure 2—figure supplement 1A. (E) Bar graph of relative FA composition by gas chromatography (GC) of methyl-esterified fatty acid extracts. These data indicate no detectable C15ISO or C17ISO was restored in elo-5(−) ku540 animals. GC graph is shown in Figure 2—figure supplement 1B. (F) and (G) Mass spectrometry of d17iso-ceramide–containing lipids by precursor scan m/z = +250.3, and d16-ceramide–containing lipids by precursor scan m/z = +236.3 (F). d17iso-ceramides and d17iso-glucosylceramides are detectable in wild-type but not elo-5(−);ku540 animals (G). In contrast, d16-ceramides and d16-glucosylceramides are present in elo-5(−);ku540, but not wild-type, animals. Numeric data for relevant peaks are shown in Figure 2—figure supplement 1C,D. (H) and (I) Percentages of animals of the indicated genotypes and treatment that reached adulthood. Error bar, SD. (H) The addition of ku540 dramatically suppressed the L1 arrest phenotype of cgt-1(−);cgt-3(RNAi) with and without the C17ISO supplement. The inclusion of elo-5(−) was due to its close linkage with ku540. When C17ISO was added to remove the negative effect of elo-5(−), about 90% of elo-5(−);ku540; cgt-1(−);cgt-3(RNAi) animals reached adulthood. C17ISO itself does not rescue the phenotype. (I). ku540 suppressed the L1 arrest phenotype the cgt-1(−);cgt-3 (−) double mutants (83.4%, n = 126). In this particular test, elo-5(−) ku540;cgt-1(−);cgt-3(−) homozygous animals were the progeny of elo-5(−/+) ku540(−/+);cgt-1(−/+);cgt-3(−) heterozygous mothers, and the presented data were obtained after normalizing against heterozygous populations (see ‘Materials and methods’). In all the other tests shown in Figure 2, elo-5(−) ku540 homozygous animals were the progeny of homozygous mothers.