Supplementary Methods
Strains
Strains were maintained as described by Brenner27 at 25°C, except for when noted. OP50
E. coli was used for feeding. The wild type reference strain was N2 Bristol. The mutant
strains used were: tub-1(nr2004)II15, and tph-1(mg280)II14. The following strains were
grown at 15°C until the L2 stage then transferred to 25°C: daf-2(e1370)III, daf2(e1370)III;daf-3(mgDf90)X, daf-16 (mgDf47)I;daf-2(e1370)III, daf-2(e1370)III;daf-18
(mg198)IV, daf-16 (mgDf47)I, daf-18 (mg198)IV, daf-3 (mgDf90)X, daf-7(e1372)III, daf7(m62)III;daf-3(mgDf90)X. Many of these strains were provided by the Caenorhabditis
Genetic Center.
Fat staining
a) Nile Red: Nile Red powder (N-1142 Molecular Probes) was dissolved in acetone at
500 g/ml, diluted in 1X phosphate buffered saline (PBS) and added on top of nematode
growth media (NGM) plates already seeded with OP50 or RNAi bacteria, to a final
concentration of 0.05 g/ml. Worms were placed on these plates as eggs or starved L1s.
Their staining phenotypes were assessed prior to starvation at the L4 and the young adult
stages. Fat content was monitored by fluorescence microscopy.
b) BODIPY-labelled fatty acids: C1-BODIPY 500/510 C12 (4,4-difluoro-5-methyl-4-bora3a,4a-diaza-s-indacene-3-dodecanoic acid), and C8-BODIPY 500/510 C5 ( 4,4-difluoro-5octoyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid) from Molecular Probes (D-3823
and D-3825) were dissolved in DMSO at 1 mg/ml. For staining animals, the stocks were
diluted 1:10,000 in 1XPBS/20 M bovine serum albumin and added on top of plates
already seeded with OP50. Worms were placed on plates as eggs or L1s. Incorporation
of BODIPY-labelled fatty acids was assessed in L4 or non-starved young adults by
fluorescence microscopy.
When co-staining, Nile Red and BODIPY-labelled fats were added to the same NGM/E.
coli plate at the indicated final concentrations.
Fluorescence microscopy, image acquisition and intensity quantitation
Nile Red and BODIPY -label fluorescence were visualized using a Zeiss SV11 M2-bio
microscope equipped with rhodamine (emission 560-590 nm) and FITC/GFP (emission
500-515 nm) filters, respectively. Images were captured using a digital CCD camera
(Hamamatsu C4742-95-12ER) attached to a Zeiss Axioplan II equipped with rhodamine
and FITC/GFP filters. All Nile Red images were acquired using identical settings and
exposure times. To quantitate pixel intensities and numbers, equal planes and regions of
the worm body were selected. Using the Openlab software (Improvision Inc. Lexington,
MA) total fluorescence for all Nile Red staining droplets within a region was calculated
as the product of area multiplied by mean fluorescence.
Lipid Analysis
L1 synchronized worms were grown on four 6-cm plates at 15°C past the L2 stage then
shifted to 25°C. Non-starved, young adult worms were washed off the plates with water,
placed into 15 ml polypropylene screw-capped centrifuge tubes, spun at 1,000 x g for 1
min, and washed 5X with water. At least 100 mg of worm pellet was required. After the
final wash, as much water as possible was removed, the sample was split into two equal
portions, and each flash frozen in dry ice/ethanol. A portion of each sample was analysed
for total soluble protein28 and the remaining pellet was used for lipid extraction. 5 ml of
ice-cold chloroform/methanol/formic acid (10:10:1) was added to the worm pellet,
vortexed for 2 min. and stored at –20°C overnight. 2.2 ml of H3PO4, 1 M KCL was
added and lipids were recovered in the chloroform phase, dried under N2 and dissolved in
0.2 ml chloroform. After the chloroform/methanol extraction the remaining worm pellet
was treated with 2.5% sulfuric acid in methanol at 80° to convert any remaining fatty
acids to fatty acid methyl esters. The hexane extract of this reaction was analysed by gas
chromatography and no fatty acids were detected. Therefore, the chloroform/methanol
extraction is >98% efficient in extracting lipids from the nematodes. Lipids were spotted
onto thin layer chromatography plates and developed in hexane-diethylether-acetic acid
(80:20:20). Lipids were located by brief staining with I2. Silica gel from the
triacylglyceride and phospholipid spots was transferred to screw capped tubes. 5 g of
15:0 was added as an internal standard and fatty acid methyl esters were prepared and
analysed by gas chromatography as previously described29.
Genome wide RNAi analysis
Bacteria containing each RNAi clone were cultured in 300 l Luria Broth media
containing 50 g/ml ampicillin for 6-14 hrs. 40 l of each culture was spotted in a single
well of a 24-well plate containing NGM agar, 6 mM IPTG and 25 g/ml carbenicillin.
After overnight incubation, Nile Red was added on top of each well to a final
concentration of 0.05 g/ml. 10 synchronized L1 worms were placed per well and
incubated at 20°C. Growth and Nile Red staining were assessed after 48, 72, and 96 hrs.
by light phase and UV microscopy. For each batch of RNAi clones tested, L4440 (vector
alone) and OP50 control wells were included. A phenotype was assigned to a well only
if a majority of the animals displayed the phenotype. All phenotypes were confirmed by
at least two additional rounds of testing on the selected clones. At the time of scoring the
Nile Red phenotype, the identities of the target RNAi clones were unknown.
Pathway analysis of the gene inactivations that cause reduced fat
For each RNAi clone, aliquots from a single RNAi culture were simultaneously tested on
wild type and all three increased fat mutant animals. Simultaneous testing of the cultures
on multiple strains insured that each RNAi clone retained its fat reducing capacity on the
wild type animals. All observations were confirmed by at least one additional round of
testing.