(A) Immunoblotting analysis of His6-tagged proteins in COS-7 cell extracts expressing His6-Hsl, His6-MmHsl, or His6-β-Gal. (B) Lipid hydrolase activities of recombinant Hsl. Cell extracts expressing …
(A) Organization of the Hsl genomic region (including the neighboring genes Ate1 and PCNA), the Hsl gene locus and the Hsl1 deletion mutant allele. Black and white boxes represent coding and …
(A) 1% agarose gel electrophoresis of diagnostic DNA products derived from genotyping PCRs of Hsl+ control and Hsl1 mutant genomic loci. DNA was amplified from fly homogenates using primers flanking …
(A) SE levels in ad libitum fed control and Hsl1 mutant flies at different times after eclosion. Data are normalized to 1-day-old (1d) control animals (n = 6). (B) SE levels in Hslb24 mutant males …
(A) Distribution of SE in body segments and tissues of adult Hsl1 mutant and control animals (n = 3). (B) Lipid droplets in steroidogenic ring glands of Hsl1 mutant and control prepupae. (C) Whole …
(A) Relative SE levels in embryos derived from reciprocal matings between Hsl1 (-/-) and control (+/+) animals at different times AEL (n = 8). (B) Free sterols in Hsl1 and control embryos at …
(A) Total levels of neutral (left panel) and polar (right panel) lipid classes during embryogenesis of control and Hsl1 animals (n = 8). (B) Combined acyl chain lengths and double bonds in major …
(A) Combined acyl chain lengths and double bonds in PG, PE O-, Cer, and CerPE (n = 8). Relative changes of individual glycerophospholipid species in control and Hsl1 animals between early (0–2 hr …
(A, B) Cumulative numbers of eggs and hatched 1st instar larvae per female on (A) LDM (n = 25–28) or (B) LDM with 0.01% cholesterol (n = 29). Note that LDM contains only traces of sterols and other …
Hatching rates of control and Hsl1 mutant embryos on (A) LDM (n = 25–28) and (B) LDM supplemented with 0.01% cholesterol (n = 29) were calculated from data depicted in Figure 7A,B. Data are …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Drosophila melanogaster) | w1118 | Vienna Drosophila Research Center (VDRC) | Cat#: 60000 | |
Genetic reagent (Drosophila melanogaster) | Hsl1 | This study | See Materials and methods | |
Genetic reagent (Drosophila melanogaster) | UAS-Hsl | This study | See Materials and methods | |
Genetic reagent (Drosophila melanogaster) | UAS-Hsl-EGFP | This study | See Materials and methods | |
Genetic reagent (Drosophila melanogaster) | bmm1 | Grönke et al., 2005 doi: 10.1016/j.cmet.2005.04.003 | FLYB: FBal0195572 | |
Genetic reagent (Drosophila melanogaster) | AkhA | Gáliková et al., 2015 doi: 10.1534/genetics.115.178897 | FLYB: FBal0319563 | |
Genetic reagent (Drosophila melanogaster) | FB-GAL4 | Grönke et al., 2003 doi: 10.1016/s0960-9822(03)00175–1 | ||
Genetic reagent (Drosophila melanogaster) | Act5c-GAL4 | Bloomington Drosophila Stock Center (BDSC) | Cat#: 4414 FLYB: FBst0004414 | backcrossed to a w1118 strain |
Genetic reagent (Drosophila melanogaster) | Cg-GAL4 | Bloomington Drosophila Stock Center (BDSC) | Cat#: 7011 FLYB: FBst0007011 | |
Genetic reagent (Drosophila melanogaster ) | nos-GAL4 | Bloomington Drosophila Stock Center (BDSC) | Cat#: 64277 FLYB: FBst0064277 | |
Genetic reagent (Drosophila melanogaster) | Hsl shRNA | Bloomington Drosophila Stock Center (BDSC) | Cat#: 65148 FLYB: FBst0065148 | |
Cell line (Chlorocebus aethiops) | COS-7 | American Type Culture Collection (ATCC) | Cat#: CRL-1651 RRID: CVCL_0224 | |
Antibody | Anti-Mouse IgG-Horseradish Peroxidase antibody; sheep | GE Healthcare | Cat#:NA931 RRID: AB_772210 | WB (1:5,000) |
Antibody | Anti-Rabbit IgG (H+L)-Horseradish Peroxidase antibody; goat polyclonal | Vector Laboratories | Cat#: PI-1000 RRID: AB_2336198 | WB (1:5,000) |
Antibody | Anti-engrailed/invected antibody; mouse monoclonal | Developmental Studies Hybridoma Bank (DSHB) | Cat#: 4D9 RRID: AB_528224 | IHC (1:100) |
Antibody | HSL antibody; rabbit polyclonal | Cell Signaling Technology | Cat#: 4107 RRID: AB_2296900 | WB (1:1,000) |
Antibody | Rabbit Anti-GAPDH antibody, unconjugated, clone 14C10; rabbit monoclonal | Cell Signaling Technology | Cat#: 2118 RRID: AB_561053 | WB (1:10,000) |
Antibody | Anti-His antibody, unconjugated; mouse monoclonal | GE Healthcare | Cat#: 27-4710-01 RRID: AB_771435 | WB 1:5000 |
Recombinant DNA reagent | pFLC-1 [Hsl] | Drosophila Genomics Resource Center (DGRC) | Cat#: RE52776 | Used for amplification of Hsl cDNA |
Transfected construct (D. melanogaster) | pcDNA4/Hismax C [His6-Hsl] | This study | See Materials and methods | |
Recombinant DNA reagent | pUAST [Hsl] | This study | See Materials and methods | |
Recombinant DNA reagent | pUAST [Hsl-EGFP] | This study | See Materials and methods | |
Sequence-based reagent | Oligonucleotides, Primers | Thermo Fisher Scientific Invitrogen | See Materials and methods | |
Commercial assay or kit | Triglycerides reagent | Thermo Fisher Scientific | Cat#: 981786 | |
Commercial assay or kit | NEFA-HR(2) | Fujifilm Wako Diagnostics | Cat#: 999–34691, Cat#: 995–34791 Cat#: 991–34891 Cat#: 993–35191 Cat#: 276–76491 | |
Commercial assay or kit | Glucose (GO) Assay kit | Sigma Aldrich | Cat#: GAGO20-1KT | |
Commercial assay or kit | RNeasy Mini kit | QIAGEN | Cat#: 74104 | |
Commercial assay or kit | QIAGEN Plasmid Midi Kit | QIAGEN | Cat#: 12143 | |
Commercial assay or kit | TRIzol | Thermo Fisher Scientific Invitrogen | Cat#: 15596026 | |
Commercial assay or kit | SuperScript III First-Strand Synthesis Supermix | Thermo Fischer Scientific Invitrogen | Cat#: 18080051 | |
Commercial assay or kit | QuantiTect Reverse Transcription Kit | QIAGEN | Cat#: 205313 | |
Commercial assay or kit | iTaq Universal SYBR Green Supermix | Bio-Rad | Cat#: 1725120 | |
Chemical compound, drug | BODIPY 493/503 | Thermo Fisher Scientific Invitrogen | Cat#: B2103 | 5 µg/ml |
Chemical compound, drug | Hoechst 33342 | Sigma Aldrich | Cat#: 14533 | 5 µg/ml |
Chemical compound, drug | CellMask Deep Red | Thermo Fisher Scientific Life Technologies | Cat#: C10046 | 1 µg/ml |
Chemical compound, drug | LipidTOX Deep Red | Thermo Fisher Scientific Life Technologies | Cat#: H3477 | (1:1,000) |
Chemical compound, drug | Lipid standards | Avanti Polar Lipids Sigma Aldrich Toronto Research Chemicals | See Materials and methods | |
Chemical compound, drug | Cholesterol | Sigma Aldrich | Cat#: C3045 | |
Chemical compound, drug | Cholesterol [25,26–3H] | American Radiolabeled Chemicals | Cat#: ART-1987 | |
Software, algorithm | LipidXplorer 1.2.7 | Herzog et al., 2012 doi:10.1371/journal.pone.0029851 | ||
Software, algorithm | Lipid Data Analyzer | Hartler et al., 2011 doi:10.1093/bioinformatics/btq699 |
Name in text/figure | Full genotype | ID/Reference |
---|---|---|
Control or +/+ | w1118; +; + | VDRC 60000 |
Hsl1 or -/- | w1118; Hsl1; + | This study |
Hslb24 | w1118; Hslb24; + | Bi et al., 2012 |
AkhA | w1118; +; AkhA | Gáliková et al., 2015 |
bmm1 | w1118; +; bmm1 | Gáliková et al., 2017 |
AkhA bmm1 | w1118; +; AkhA bmm1 | This study |
Hsl1 bmm1 | w1118; Hsl1; bmm1 | This study |
Hsl1 AkhA | w1118; Hsl1; AkhA | This study |
UAS-Hsl or +/+; UAS-Hsl | w1118; +; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+ | This study |
Act5c> | w*; P{w+mC=Act5C-GAL4}25FO1/+; + | BDSC 4414 |
Act5c>UAS-Hsl | w*; P{w+mC=Act5C-GAL4}25FO1/+; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+ | This study, BDSC 4414 |
Hsl1 UAS-Hsl or -/- UAS-Hsl | w1118; Hsl1; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+ | This study |
Hsl1 Act5c | w*; Hsl1 P{w+mC=Act5C-GAL4}25FO1/Hsl1; + | This study, BDSC 4414 |
Hsl1 Act5c UAS-Hsl | w*; Hsl1 P{w+mC=Act5C-GAL4}25FO1/Hsl1; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+ | This study, BDSC 4414 |
FB>+ or FB-GAL4 | w*; P{w+mW.hs=GawB}FB/+; + | Grönke et al., 2003 |
Hsl1 FB | w*; Hsl1 P{w+mW.hs=GawB}FB/Hsl1; + | This study |
Hsl1 FB UAS-Hsl | w*; Hsl1 P{w+mW.hs=GawB}FB/Hsl1; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+ | This study |
Hsl1 Myo31DF | w*; Hsl1 P{w+mW.hs=GawB}Myo31DFNP0001/Hsl1; + | This study, BDSC 67088 |
Hsl1 Myo31DF UAS-Hsl | w*; Hsl1 P{w+mW.hs=GawB}Myo31DFNP0001/Hsl1; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+ | This study, BDSC 67088 |
Hsl1 Desat1 | w*; Hsl1 P{w+mC=Desat1-GAL4.E800}2M/Hsl1; + | This study, BDSC 65404 |
Hsl1 Desat1 UAS-Hsl | w*; Hsl1 P{w+mC=Desat1-GAL4.E800}2M/Hsl1; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+ | This study, BDSC 65404 |
Hsl1 elav | w*; Hsl1; P{w+mC=GAL4-elav.L}3/+ | This study, BDSC 8760 |
Hsl1 elav UAS-Hsl | w*; Hsl1; P{w+mC=GAL4-elav.L}3/ P{w+mC Hsl [Scer\UAS]=UAS-Hsl} | This study, BDSC 8760 |
-/-; nos-GAL4 | w*; Hsl1; P{w+mC=GAL4::VP16-nos.UTR}1C/+ | This study, BDSC 64277 |
-/-; nos-GAL4/UAS-Hsl | w*; Hsl1; P{w+mC=GAL4::VP16-nos.UTR}1C/ P{w+mC Hsl [Scer\UAS]=UAS-Hsl} | This study, BDSC 64277 |
Cg>+ | w[1118]/y[1] v[1]; P{w[+mC]=Cg-GAL4.A}2/ P{y[+t7.7]=CaryP}attP40; + | BDSC 7011 and BDSC 36304 |
Hsl shRNA/+ | w[1118]/y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMC05951}attP40/+; + | BDSC 65148 and VDRC 60000 |
Cg>Hsl shRNA | w[1118]/y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMC05951}attP40/ P{w[+mC]=Cg-GAL4.A}2; + | BDSC 7011 and BDSC 65148 |
Gene symbol | Primer sequences | ID/Reference |
---|---|---|
rp49/RpL32 | fw: 5‘-CTTCATCCGCCACCAGTC-3‘ rv: 5‘-CGACGCACTCTGTTGTCG-3‘ | |
plin1 | fw: 5‘-GCGCGAATTCTGGCGCCCCTAGATG-3‘ rv: 5‘-CACAGAAGTAAGGTTCCTTCACAAAGATCC-3‘ | |
plin2 | fw: 5’-TCAAATTGCCCGTGGTAAA-3’ rv: 5’-CCCATTCGAAGACACGATTT-3’ | |
Akh | - | QIAGEN QuantiTect QT00957859 |
bmm | - | QIAGEN QuantiTect QT00964460 |
Hr96 | fw: 5’-CCAGCGAGGCTCTTTATGAT-3’ rv: 5’-GGTTGTGGCGAGTGTCGT-3’ | |
Npc1a | fw: 5’-GTCGAGGAACTTTGCAGGGA-3’ rv: 5’-TCATCGAAACAGGACTGCGT-3’ | |
Npc1b | fw: 5’-CGGATTTTGTTCCAGCAACT-3’ rv: 5’-CCATTCTCAGTAAATCCTCGTTC-3’ | |
Npc2a | fw: 5’-ACAGTCGTCCACGGCAAG-3’ fw: 5’-ACACAGGCATCGGGATTG-3’ | |
Npc2b | fw: 5‘-GGAGATCCACTGGGGATTG-3‘ rv: 5‘-CCTTGATTTTGGCGGGTAT-3‘ | |
CG8112 | fw: 5’-CACAAACTGAAACCGCACAG-3’ rv: 5’-CGACACGAAACAGAAGACCA-3’ | |
magro | fw: 5’-ACACCGAACTGATTCCGAAC-3’ rv: 5’-ATCCACCATTGGCAAACATT-3’ | |
Hsl | fw: 5‘-CTGGAGGCGACCTATGGAAC-3‘ rv: 5‘-GCTCGTCAAAATCGTACTCGTG-3‘ | |
PCNA | fw: 5‘-GCGACCGCAATCTCTCCAT-3‘ rv: 5‘-CGCCTTCATCGTCACATTGT-3‘ | |
Ate1 | fw: 5‘-GCATACTTCGCCGCATAAATCG-3‘ rv: 5‘-CTATGGCGTAATCGGCATCGG-3‘ | |
MmAbca1 | fw: 5’-GATGTGGAATCGTCCCTCAGTTC-3’ rv: 5’-ACTGCTCTGAGAAACACTGTCCTCC-3’ | |
MmSoat1 | fw: 5’-GAAACCGGCTGTCAAAATCTGGR-3’ rv: 5’-TGTGACCATTTCTGTATGTGTCC-3’ | |
MmHmgcs1 | fw: 5’-GACAAGAAGCCTGCTGCCATA-3’ rv: 5’-CGGCTTCACAAACCACAGTCT-3’ | |
MmHmgcr | fw: 5’-TGCACGGATCGTGAAGACA-3’ rv: 5’-GTCTCTCCATCAGTTTCTGAACCA-3’ | |
MmSrebp2 | fw: 5’-GCGCCAGGAGAACATGGT-3’ rv: 5’-CGATGCCCTTCAGGAGCTT-3’ | |
MmStar | fw: 5’-TTGGGCATACTCAACAACCA-3’ rv: 5’-GAAACACCTTGCCCACATCT-3’ | |
Mm36B4 | fw: 5’-GCTTCATTGTGGGAGCAGACA-3’ rv: 5’-CATGGTGTTCTTGCCCATCAG-3’ |
Composition of the lipidome.