<i>dhs-16</i> mutant animals are deficient in lathosterone.

<p>(A) <i>daf-9(k182)</i> rescue is seen only with the DAs. Experiments were carried out at 27°C with 33 µM concentration of supplemented compounds (<i>N</i>≥3, M ± SD; ***<i>p</i><0.0001). (B) <i>dhs-16(tm1890)</i> rescue is seen with lathosterone and dafachronic acids, but not by cholesterol, 7-dehydrocholesterol, and lathosterol. Similarly, 4-cholesten-3-one rescues, but not cholesterol or 4-cholesten-3β-ol. This indicates that <i>dhs-16</i> may function in the conversion of lathosterol to lathosterone in the production of Δ⁷-DA and in the formation of 4-cholestene-3-one in the production of Δ⁴-DA (N≥3, M ± SD; ***<i>p</i><0.0001). (C) <i>emb-8</i> RNAi rescue is seen only with the DAs, and not with lathosterone or 4-cholesten-3-one. (<i>N</i>≥3, M ± SD; **<i>p</i><0.01). (D) LC/MS/MS analysis of lipid extracts from L3 stage animals reveals that lathosterone levels are reduced in <i>dhs-16(tm1890)</i> mutant animals compared to N2 wild-type (WT) animals, shown quantitatively in (E). Lathosterone levels are significantly reduced in <i>dhs-16</i> animals relative to N2 wild-type (3.5-fold decrease; <i>N</i> = 7, M ± SD; **<i>p</i><0.001). (F) GC/MS/MS analysis of sterol levels in <i>dhs-16</i> mutants reveals deficiencies in lathosterone, Δ⁷-dafachronic acid, and 4-methyl sterols compared to N2 wild-type (<i>N</i>≥10, M ± SEM; *<i>p</i><0.05, **below detection limit).