Supplemental Methods.
Worm lysis, immunoblots, and preparation of anti-MDT-15 polyclonal antibody,
N2 worms were collected and washed 5 times in M9, flash frozen in liquid N2, thawed in lysis buffer (150mM KCl, 1mM EDTA, 0.25% SDS, 1.0% NP-40, 50mM Tris/HCl pH7.4, and Roche Complete protease inhibitors), sonicated, and cleared by centrifugation. Equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane (Millipore #IPVH00010), and subsequently analyzed by standard immunoblot techniques. An N-terminal peptide of MDT-15 [MSEEDWPSPKFREHVIQRLEPEC; BLASTP search against the C. elegans proteome revealed no similarities to proteins other than MDT-15, p-value >0.0001 (data not shown)] was used to raise polyclonal antisera in rabbits (Covance, Berkeley CA). Some peptide was coupled to Sulfolink resin (PIERCE # 20405) according to the manufacturer’s protocol; this affinity matrix was then used to affinity-purify the crude rabbit bleeds. The resulting affinity purified anti-MDT-15 antibody was used for immunoblots at a concentration of 0.5g/ml. The anti-actin monoclonal antibody was from Chemicon (MAB1501R).
Supplemental Table S1. Summary of fatty acid metabolism gene expression data in N2 L4 worms. Expression data were collected as Ct values, where Ct is equal to the number of PCR cycles required to amplify a given gene from a cDNA population. Changes in gene expression between control(RNAi) and mdt-15(RNAi) worms are represented as Ct values; thus, Ct = Ct[control(RNAi)] – Ct[mdt-15(RNAi)] Each value represents averages of duplicate qRT-PCR data from three independent experiments; SEM = standard error of the mean.
Acc. No. / Gene / Function / Ct (control RNAi -mdt-15 RNAi) / SEM
R06F6.9 / ech-4 / Enoyl-CoA Hydratase / 0.312 / 0.04
K04F1.15 / alh-2 / Aldehyde Dehydrogenase / -0.274 / 0.14
C29F3.1 / ech-1 / Enoyl-CoA Hydratase / -1.168 / 0.12
F38H4.8 / ech-2 / Enoyl-CoA Hydratase / 0.046 / 0.03
F43H9.1 / ech-3 / Enoyl-CoA Hydratase / 1.059 / 0.14
F56B3.5 / ech-5 / Enoyl-CoA Hydratase / -0.145 / 0.06
T05G5.6 / ech-6 / Enoyl-CoA Hydratase / -0.912 / 0.08
Y105E8A.4 / ech-7 / Enoyl-CoA Hydratase / 1.157 / 0.13
T08B2.7 / 3-Hydroxyacyl-CoA Dehydrogenase / -1.464 / 0.33
F37C12.7 / LC FA-CoA Ligase / 0.821 / 0.17
R07C3.4 / LC FA-CoA Ligase / 0.912 / 0.30
R09E10.3 / LC FA-CoA Ligase / -0.206 / 0.73
R09E10.4 / LC FA-CoA Ligase / 0.980 / 0.11
Y65B4BL.5 / LC FA-CoA Ligase / -0.247 / 0.14
Y76A2B.3 / LC FA-CoA Ligase / -0.919 / 0.09
T08B2.7 / 3-Hydroxyacyl-CoA Dehydrogenase / 0.104 / 0.13
B0272.3 / 3-Hydroxyacyl-CoA Dehydrogenase / -0.357 / 0.38
B0303.3 / Thiolase / 0.227 / 0.12
C07E3.9 / Phospholipase / 1.387 / 0.14
C25A1.5 / VLC FA Hydoxylase / 0.322 / 0.18
C42D8.5 / acn-1 / Angiotensin Converting Enzyme / 0.136 / 0.37
C44B7.8 / pmp-1 / ATP-Binding Protein (peroxisome) / 0.244 / 0.16
C44B7.9 / pmp-2 / ATP-Binding Protein (peroxisome) / -0.357 / 1.43
C48B4.1 / Acyl-CoA Oxidase / 1.421 / 0.27
C50D2.7 / GlucoKinase / 1.072 / 0.42
F25C8.1 / Acyl-CoA Oxidase / -1.011 / 0.20
EEED8.3 / Cytosolic FA Binding / 0.175 / 0.16
F01G10.3 / ech-9 / Enoyl-CoA Hydratase / 2.167 / 0.35
F01G4.2 / ard-1 / 3-Hydroxyacyl-CoA Dehydrogenase / -0.049 / 0.13
F08A8.1 / Acyl-CoA Oxidase / -1.832 / 0.06
F08A8.2 / Acyl-CoA Oxidase / -6.712 / 1.36
F08A8.3 / Acyl-CoA Oxidase / -3.171 / 0.43
F08A8.4 / Acyl-CoA Oxidase / -2.268 / 0.06
F33D4.4 / FA Desaturase / 0.393 / 0.16
F40F4.2 / lbp-2 / Lipid-binding protein / 0.964 / 0.17
F40F4.3 / lbp-1 / Lipid-binding protein / -0.074 / 0.20
F40F4.4 / lbp-3 / Lipid-binding protein / 0.684 / 0.21
F53A2.7 / Thiolase / -0.458 / 0.14
F59F4.1 / Acyl-CoA Oxidase / -0.233 / 0.11
H12D21.5 / Thiosulfate Sulfurtransferase / 0.272 / 0.27
K05F1.3 / Acyl-CoA Dehydrogenase / 0.621 / 0.19
T02G5.4 / Acetyl-CoA Thiolase / -0.522 / 0.11
T02G5.7 / Acetyltransferase / -0.430 / 0.10
T02G5.8 / kat-1 / Acetyl-CoA acetyltransferase / -0.559 / 0.09
T05E7.3 / Cytosolic fatty-acid binding protein / -0.228 / 1.28
T12F5.3 / glh-4 / ATP-dependent RNA helicase / 0.668 / 0.06
T22G5.2 / lbp-7 / Lipid-binding protein / -1.876 / 0.07
T22G5.6 / lbp-8 / Lipid-binding protein / -9.891 / 0.06
T25G12.5 / 3-Hydroxyacyl-CoA Dehydrogenase / 0.152 / 0.05
VZK822L.1 / fat-6 / FA-9-desaturase / -4.857 / 0.16
W02D3.5 / lbp-6 / Lipid-binding protein / 0.104 / 0.12
W02D3.7 / lbp-5 / Lipid-binding protein / -0.922 / 0.14
W06D12.3 / fat-5 / FA desaturase / -4.210 / 0.09
W09B6.1 / phi-47 / Acetyl-CoA Carboxylase / 0.392 / 0.09
F28F8.2 / acs-2 / LC FA-CoA Ligase / -0.769 / 0.25
F10D2.9 / fat-7 / FA-9-desaturase / -8.835 / 0.13
F13D12.9 / Phospholipid Biosynthesis / 0.663 / 0.23
Y39G10AR.2 / zwl-1 / Cytosolic FA binding protein / 0.308 / 0.05
Y40B10A.1 / lbp-9 / Cytoplasmic FA-binding protein FABP / 0.686 / 0.15
Y54E5A.1 / FA Desaturase / 1.051 / 0.17
Y56A3A.19 / Acyl carrier protein / 0.099 / 0.12
ZK742.5 / lbp-4 / FA-binding protein FABP / 0.008 / 0.29
C15F1.7 / sod-1 / Cu2+/Zn2+ superoxide dismutase / 0.193 / 0.06
F10D11.1 / sod-2 / Manganese superoxide dismutase / 0.333 / 0.17
C08A9.1 / sod-3 / Manganese superoxide dismutase / -0.897 / 0.05
F55H2.1 / sod-4 / Cu2+/Zn2+ superoxide dismutase / 1.643 / 0.29
C46C11.1 / Hormone Sensitive Lipase / 0.690 / 0.11
F11E6.5 / elo-2 / Palmitic Acid Elongase / 0.062 / 0.14
F47F2.1 / PKA catalytic subunit / 0.255 / 0.20
Y54G11A.5 / ctl-2 / Catalase / -0.236 / 0.24
Y54G11A.13 / ctl-3 / Catalase / -0.198 / 0.45
C02B10.1 / Isovaleryl-CoA dehydrogenase (SC-ACD) / -0.870 / 0.08
R09B5.6 / hacd-1 / 3-OH-Acyl-CoA-DH / 1.525 / 0.16
F28D1.9 / VL-ACS / 0.952 / 0.13
F54C8.1 / 3-OH-Acyl-CoA-DH / 0.740 / 0.14
T13F2.1 / fat-4 / FA-5-desaturase / 0.228 / 0.12
E04F6.5 / VLC-ACD / -0.128 / 0.14
C55B7.4A / acdh-1 / Short-chain acyl-CoA dehydrogenase / -7.763 / 0.87
C17C3.12A / acdh-2 / Short-chain acyl-CoA dehydrogenase / -3.560 / 0.09
F41C3.3 / acs-11 / Acyl-CoA synthetase / 0.243 / 0.11
F41H10.7 / elo-5 / Long chain FA elongase / 0.718 / 0.04
F41H10.8 / elo-6 / Long chain FA elongase / -1.263 / 0.18
F56H11.3 / elo-7 / Long chain FA elongase / 1.129 / 0.11
Y47D3A.30 / elo-8 / Long chain FA elongase / -0.565 / 0.81
Y53F4B.2 / elo-9 / Long chain FA elongase / -0.277 / 0.43
K07B1.3 / ucp-4 / Mitochondrial FA anion carrier protein / -0.178 / 0.18
F32H2.5 / Animal-type FA synthase / 0.022 / 0.01
Y67H2A.8 / fat-1 / Oleate desaturase/linoleate desaturase / -0.940 / 0.11
W02A2.1 / fat-2 / Oleate desaturase/linoleate desaturase / -1.867 / 0.11
W08D2.4 / fat-3 / FA-6-desaturase / 0.021 / 0.05
Y46G5A.17 / cpt-1 / Carnitine O-acyltransferase CPT1 / -0.075 / 0.05
R07H5.2 / cpt-2 / Carnitine O-acyltransferase CPT2/YAT1 / 0.236 / 0.05
T20B3.1 / Carnitine O-acyltransferase CROT / -0.162 / 0.20
F09F3.9 / cpt-5 / Carnitine O-acyltransferase CPT1 / -5.718 / 0.45
Y48G9A.10 / cpt-3 / Carnitine O-acyltransferase CPT1 / -0.168 / 0.14
W01A11.5 / cpt-6 / Carnitine O-acyltransferase CPT1 / -0.612 / 0.11
F41E7.6 / Carnitine O-acyltransferase CROT / 0.528 / 0.23
B0395.3 / Carnitine O-acyltransferase CRAT / -0.046 / 0.06
C01H6.5 / nhr-23 / Nuclear Hormone Receptor / 0.234 / 0.10
F36A4.7 / ama-1 / RNA polymerase II, large subunit / 0.519 / 0.05
Total: / 96
Up > 4 / 1
Up 2 to 4 / 8
Between -2 and +2 / 72
Down -2 to -4 / 6
Down < -2 / 9
Supplemental Table S2. Summary of glucose metabolism gene expression data in N2 L4 worms. Expression data were collected as Ct values, where Ct is equal to the number of PCR cycles required to amplify a given gene from a cDNA population. Changes in gene expression between control(RNAi) and mdt-15(RNAi) worms are represented as Ct values; thus, Ct = Ct[control(RNAi)] – Ct[mdt-15(RNAi)] Each value represents averages of duplicate qRT-PCR data from three independent experiments; SEM = standard error of the mean.
Acc. No. / Gene / Function / Ct (control RNAi -mdt-15 RNAi / SEM
C05C10.3 / Succinyl-CoA-3-ketoacid CoA Transferase / -1.438 / 0.12
F25B4.6 / HMG-CoA Synthase / 0.625 / 0.07
Y71G12B.10 / HMG-CoA Lyase / 0.173 / 0.09
F14B4.2 / Hexokinase / -0.036 / 0.15
H25P06.1 / Hexokinase / 0.013 / 0.03
Y71H10A.1 / 6-Phosphofructokinase (both spliceforms) / 1.995 / 1.37
C50F4.2 / 6-phosphofructokinase / 0.348 / 0.39
R11A5.4 / PEPCK (all spliceforms) / -1.003 / 0.16
W05G11.6 / PEPCK (all spliceforms) / 0.223 / 0.15
F25H5.3 / Pyruvate Kinase (all spliceforms) / -0.183 / 0.05
ZK593.1 / Pyruvate Kinase / 0.318 / 0.16
Y110A7A.6 / Phosphofructokinase (both spliceforms) / 0.417 / 0.11
K02B2.1 / Phosphofructokinase / 0.283 / 0.02
H17B01.1 / Sugar Transporter (both spliceforms) / 0.528 / 0.07
R09B5.11 / Sugar Transporter / 1.304 / 0.26
K10B3.7 / gpd-3 / Glyceraldehyde-3 Phosphate DH / -0.099 / 0.10
K10B3.8 / gpd-2 / Glyceraldehyde-3 Phosphate DH / -0.376 / 0.09
T09F3.3 / gpd-1 / Glyceraldehyde-3 Phosphate DH / -0.061 / 0.09
F33H1.2 / gpd-4 / Glyceraldehyde-3 Phosphate DH / 0.221 / 0.07
R11A5.4 / PEPCK (a and b spliceforms) / -1.302 / 0.09
W05G11.6 / PEPCK (a and d spliceforms) / -0.019 / 0.14
W05G11.6 / PEPCK (a,b and d spliceforms) / 0.644 / 0.13
W05G11.6 / PEPCK (a,b and c spliceforms) / 1.404 / 0.13
F25H5.3 / Pyruvate Kinase (b spliceform) / 0.925 / 0.12
F25H5.3 / Pyruvate Kinase (a and b spliceforms) / 0.075 / 0.11
Y110A7A.6 / Phosphofructokinase (a isoform) / 0.258 / 0.11
H17B01.1 / Sugar Transporter (b isoform) / 0.479 / 0.11
K07A3.1 / fbp-1 / Fructose, 1,6 bisphosphatase / -1.038 / 0.07
F54H12.1 / aco-2 / Aconitase (a and b spliceforms) / -0.256 / 0.07
F54H12.1 / aco-2 / Aconitase (a, b and c spliceforms) / 0.084 / 0.15
ZK455.1 / aco-1 / Aconitase / -0.474 / 0.06
F20H11.3 / mdh-1 / Malate dehydrogenase / -0.711 / 0.06
F46E10.10 / Lactate/malate dehydrogenase (a and c) / -0.399 / 0.11
F46E10.10 / Lactate/malate dehydrogenase (a and b) / -0.708 / 0.07
C05E4.9 / gei-7 / Isocitrate lyase family/Malate synthase / -0.343 / 0.15
C05E4.9 / gei-7 / Isocitrate lyase family/Malate synthase / -0.547 / 0.16
F48E8.3 / Fumarate Reductase / 1.512 / 0.15
C03G5.1 / Succinate dehydrogenase / -0.436 / 0.05
C34B2.7 / Succinate dehydrogenase / 0.186 / 0.07
R11F4.1 / Glycerol kinase / 0.448 / 0.15
F47G4.3 / NAD-GAPDH / -1.577 / 0.12
Y71H10A.1 / 6-phosphofructokinase (a spliceform) / 0.592 / 0.16
R11A5.4 / PEPCK (a, c and d isoforms) / -1.012 / 0.07
Total: / 43
Up > 4 / 0
Up 2 to 4 / 3
Between -2 and +2 / 34
Down -2 to -4 / 6
Down < -2 / 0
Supplemental Table S3. Summary of DAF-12 target gene expression data in N2 L4 worms. Expression data were collected as Ct values, where Ct is equal to the number of PCR cycles required to amplify a given gene from a cDNA population. Changes in gene expression between control(RNAi) and mdt-15(RNAi) worms are represented as Ct values; thus, Ct = Ct[control(RNAi)] – Ct[mdt-15(RNAi)] Each value represents averages of duplicate qRT-PCR data from three independent experiments; SEM = standard error of the mean.
Acc. No. / Gene / Function / Ct (control RNAi -mdt-15 RNAi) / SEM
ZC404.1 / 0.538 / 0.16
C46E1.1 / -0.196 / 0.12
C46E1.2 / gcy-36 / Adenylate/guanylate kinase / 0.397 / 0.21
T06A1.1 / Extracellular protein with conserved cysteines / 0.414 / 0.36
F27C1.10 / -0.926 / 0.29
C37C3.3 / 0.589 / 0.32
C37C3.8 / tag-253 / Predicted dioxygenase / -0.125 / 0.15
C32A3.1 / sel-8 / 0.066 / 0.12
T13F2.8 / cav-1 / Caveolin / -0.759 / 0.37
W06F12.2 / 0.875 / 0.27
F28E10.1 / -0.339 / 0.15
W10G6.3 / ifa-2 / Nuclear envelope protein lamin / -0.997 / 0.19
T06A1.5 / Extracellular protein with conserved cysteines / 0.388 / 0.19
F08C6.1 / adt-2 / Disintegrin metalloproteinase / -0.391 / 0.08
F08C6.2 / Phosphorylcholine transferase / 0.235 / 0.14
C28A5.3 / nex-3 / Annexin / 0.063 / 0.25
C28A5.2 / 0.116 / 0.06
F28D1.10 / gex-3 / Membrane-associated hematopoietic protein / -0.227 / 0.14
F08G5.5 / UDP-glucuronosyl and UDP-glucosyl transferase / -0.167 / 0.39
K11C4.2 / Predicted membrane protein / 0.156 / 0.08
K11C4.1 / Casein kinase / -2.464 / 0.08
K11C4.4 / odc-1 / Ornithine decarboxylase / 0.674 / 0.23
Y19D10A.8 / -0.951 / 0.07
Y19D10A.12 / -0.352 / 0.07
Y19D10A.5 / Permease of the major facilitator superfamily / -2.325 / 0.67
Y19D10A.4 / Predicted mutarotase / -0.353 / 0.19
T07H6.2 / mom-1 / Predicted acyltransferase / -0.198 / 0.16
W06F12.1 / lit-1a / Nemo-like MAPK-related kinase / 1.549 / 0.20
W06F12.1 / lit-1b / 0.085 / 0.11
W06F12.1 / lit-1 / 0.966 / 0.12
Total: / 30
Up > 4 / 0
Up 2 to 4 / 1
Between -2 and +2 / 27
Down -2 to -4 / 0
Down < -2 / 2
Supplemental figure legends.
Figure S1. The promoter of mdt-15 drives intestinal and neuronal expression at all larval stages.
DIC and GFP micrographs of BC11928 worms at individual larval stages. The promoter of mdt-15 drives intestinal and neuronal GFP-expression at larval stages L1 (top; scale bar represents 32.5 m), L2 and L3 (middle; scale bar represents 32.5 m), and L4 (bottom; scale bar represents 48.6 m). Pha = pharynx, Neu = head neurons, Vul = vulva.
Figure S2. mdt-15 RNAi results in efficient and specific knockdown of MDT-15 mRNA and protein.
(A) QRT-PCR quantification of relative mRNA levels in worms grown on control RNAi (blue), nhr-49 RNAi (red), mdt-15 RNAi (yellow), or mdt-6 RNAi (green). Each bar represents the average relative mRNA level from three independent RNA isolations (normalized to ama-1 mRNA levels) from N2 L4 stage animals; error bars represent SEM. (B) Bar graphs represent fold reduction of indicated mRNA levels after different RNAi treatments (relative to control RNAi); mdt-6 RNAi does affect lbp-8 and fat-7 levels, but exhibits only minor impact on levels of fat-5 and fat-6 mRNA. (C) Immunoblot analysis of whole-worm lysates from N2 L4 worms grown on different RNAi bacteria, as indicated. mdt-15 RNAi, but neither nhr-49 nor mdt-6 RNAi, strongly reduced MDT-15 protein level, while -actin levels are unaffected. One of three independent experiments with similar outcome is shown.