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. Author manuscript; available in PMC: 2013 Dec 1.
Published in final edited form as: Nat Cell Biol. 2013 Apr 21;15(6):668–676. doi: 10.1038/ncb2741

Figure 3.

Figure 3

MXL-3 represses lysosomal lipolysis in ad libitum-fed conditions. (a) Expression of lipl genes in well-fed mxl-3(ok1947) and mxl-3(tm2580) young adults normalized to same age well-fed wild-type (WT) worms shows that mxl-3 loss of function is sufficient to induce lipl-1 to 3 and lipl-5. Mean ddCts+ s.e.m. are depicted. n = 6 independent experiments for lipl-1 to lipl-5, and n = 4 independent experiments for lipl-6 to lipl-8. (b) L4 larvae were fasted and RNA was extracted at the indicated times. Mean ddCts+ s.e.m. show that the response mxl-3 orchestrates is transient. Time 0, n = 6 independent experiments; time 3–12 h, n = 4 independent experiments; time 18–24 h, n = 2 independent experiments. (c) Immunostainings of well-fed or 6 or 12 h fasted MXL-3::GFP young adults are presented. Quantification of GFP-positive nuclei relative to total intestinal nuclei (4,6-diamidino-2-phenylindole, DAPI) of two independent experiments shows that MXL-3 transiently delocalizes from the intestinal nuclei during fasting. (d) ChIP–quantitative PCR (ChIP-qPCR) analysis of well-fed and 6 h fasted mixed-stage worms expressing MXL-3::GFP presented as Ct in αGFP immunoprecipitated DNA normalized to input DNA and relative to a mock promoter region (CACTAT site −88 of ama-1 gene) shows that MXL-3 vacates the lipl promoters during early fasting. Three sets of primers surrounding CACGTG target sites found up to 500 bp of the ATG of the lipl-1 and lipl-3 genes were used. A representative experiment is presented; see raw data of two independent experiments in Supplementary Table S9. (e) Wild-type or lipl-1(tm1954) lipl-3(tm4498) double mutant animals grown on control RNAi plates were transferred as L4 larvae to control or mxl-3 RNAi plates, incubated for 12 h at 20 °C, and processed for total FAMEs; as a reference, an aliquot of wild-type young adults on control RNAi bacteria was fasted for 6 h. FAME quantification (mean percentage of fed wild type ±s.e.m.) shows that acute mxl-3 inactivation, as fasting, reduces fat stores in a lipl-dependent manner. n = 3 independent experiments. NSD, P-value > 0.05.