Figures and data in Hormone-sensitive lipase couples intergenerational sterol metabolism to reproductive success

Figures
Tables
Additional files

7 figures, 3 tables and 2 additional files

Figures

Figure 1

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Enzyme activities and subcellular localization of *Drosophila* Hsl.

(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 His₆-Hsl or His₆-MmHsl were incubated with different lipids, the release of FAs was measured and normalized to the His₆-β-Gal control (n = 3). (C) Lipid hydrolase activities of *Hsl* gain-of-function flies. *UAS-Hsl* transgene expression was ubiquitously driven in vivo using the *Act5c-GAL4* driver. Lipid hydrolase activities of abdominal extracts were determined as in (B). Flies harboring either the *Act5c-GAL4* transgene or the non-induced *UAS-Hsl* transgene only served as controls and data were normalized to *Act5c-GAL4>+* samples (n = 4). (D) Lipid droplet-associated localization of Hsl-EGFP in fat body cells. Fat body tissue expressing a *UAS-Hsl-EGFP* transgene driven by *Act5c-GAL4* was stained with LipidTOX Deep Red to detect lipid droplets and imaged by confocal fluorescence microscopy. Scale bar: 10 µm. All data are presented as means and SD. Statistical significance was determined by (B) one-way ANOVA (a, p>0.05 vs His₆-β-Gal) and (C) one-way ANOVA (a, p<0.05 *Act5c>UAS-Hsl* vs. *UAS-Hsl*/+ and b, p<0.05 *Act5c*>*UAS-Hsl* vs *Act5c*>+).

Figure 2 with 1 supplement

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Normal TG and energy metabolism in *Hsl¹* mutant flies.

(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 non-coding exons, respectively. Residual P-element sequences are indicated by a grey triangle. (B) Abdominal fat body tissue of ad libitum fed control and *Hsl¹* mutant animals was stained with Hoechst 33342, Cellmask Deep Red and BODIPY 493/503 to visualize nuclei, cell membranes and LDs, respectively, and imaged by confocal fluorescence microscopy. Scale bars: 10 µm. (C) Whole-body TG and DG levels of ad libitum fed *Hsl¹* mutant and control animals as determined by shotgun MS (n = 7–8). (D) TG composition of ad libitum fed *Hsl¹* and control animals as determined by UPLC-MS (n = 4). (E) Lipolytic gene expression in *Hsl¹* mutants. Relative mRNA concentrations were determined by qPCR and normalized to controls (n = 4). (F) TG equivalents of ad libitum fed and starved *Drosophila* mutants. Flies were fed ad libitum for 7 days and TG equivalents were determined by a coupled colorimetric assay either before or after starvation to death (n = 4). (G) Metabolite levels in ad libitum fed *Hsl¹* mutant and control flies were measured by colorimetric assays (n = 8). (H) Heat dissipation of ad libitum fed or starved *Hsl¹* mutant and control flies was determined by microcalorimetry (n = 5). (I) Starvation sensitivity of *Hsl¹* mutant flies. 7-days-old flies were subjected to starvation and survival was monitored every 2–12 hr (n = 39–40). Data are presented as mean and SD (**C, D, E, F, G, H**) or Kaplan-Meier curve (I). Statistical significance was determined by (**C, D, E, G, H**) unpaired t-tests (#, p>0.05 compared to control), (F) one-way ANOVA (a, p<0.05 compared to ad libitum fed control; b, p<0.05 compared to starved control) and (I) log-rank test.

Figure 2—figure supplement 1

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Molecular characterization of the *Hsl¹* mutant allele.

(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 the deleted region of the *Hsl¹* allele. (B) Relative mRNA levels of *Hsl*, *Ate1*, and *PCNA* were determined by qPCR and are normalized to controls (n = 4). Data are presented as mean and SD. Statistical significance was determined by unpaired t-tests (#, p<0.05).

Figure 3

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Sterol metabolism in *Hsl¹* mutant flies.

(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 *Hsl^b24* mutant males and females 7 days after eclosion. Data are normalized to controls (n = 3). (C) Normalized neutral SE hydrolase activities of *Hsl¹* mutant and control abdominal extracts (n = 8). (**D–F**) Turnover of radiolabeled sterols in *Hsl¹* mutant animals. Larvae were reared on food containing (D) ¹⁴C-FA or (E) ³H-cholesterol, switched to non-labeled food after eclosion and radioactivity in (**D, E**) SE, and (F) free sterol fractions was determined at the indicated timepoints (n = 4–5). Red and black colors in chemical structures indicate radiolabeled and unlabeled lipid moieties, respectively. (G) Non-esterified sterols in ad libitum fed control and *Hsl¹* mutant animals as determined by shotgun MS (n = 7–8). (H) Relative mRNA levels of genes involved in sterol transport and metabolism. Relative mRNA levels were determined by qPCR and normalized to controls (n = 4). Data are presented as mean and SD. Statistical significance was determined by unpaired t-tests (#, p<0.05).

Figure 4

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Adipocyte-autonomous control of organismal SE by Hsl.

(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 body SE after *GAL4*-driven re-expression of *UAS-Hsl* in all tissues (*Act5c*), fat body (FB), intestine (*Myo31DF*), oenocytes (*Desat1*), or nervous system (*elav*) of *Hsl¹* mutant animals. Levels are normalized to controls (n = 3). (D) Relative whole-body SE levels after fat body-specific expression of Hsl in control animals (n = 3). (E) Immunoblotting analysis of HSL in tissues of mice with adipocyte-specific disruption of the *HSL* gene (AHKO) and controls. GAPDH was used as a control. (F) Relative TG and SE levels in SCAT, PGAT, and PRAT of AHKO and control mice (n = 3–5). (G) SE hydrolase activities in soluble PGAT extracts of control and AHKO mice (n = 4). (H) Relative mRNA levels of sterol metabolism genes in PGAT of control and AHKO mice. mRNA levels were measured by qPCR and are normalized to controls (n = 4–5). Data are presented as means and SD. Statistical significance was determined by (**A, F, G, H**) unpaired t-tests (#, p<0.05) and (**C, D**) one-way ANOVA (a, p<0.05 compared to control; b, p<0.05 compared to control + *UAS-Hsl*).

Figure 5

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Maternal Hsl determines embryonic sterol homeostasis.

(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 different times AEL (n = 8). (C) Relative SE hydrolysis rates and SE levels in control and *Hsl¹* mutant embryos and mothers after *nos-GAL4-*driven re-expression of *UAS-Hsl* in the germline (n = 3). (D) Hatching rates of control and *Hsl¹* mutant embryos (n = 5). (E) Heat dissipation rates of control and *Hsl¹* mutant embryos as determined by microcalorimetry (n = 12). (F) Immunohistochemical detection of Engrailed in control and *Hsl¹* mutant embryos at different times AEL. Scale bar: 50 µm. Data are presented as means and SD. Statistical significance was determined by (**B, D**) unpaired t-tests (#, p<0.05), (A) one-way ANOVA (a, b, c, p<0.05 vs +/+ at 0–2 hr, 6–8 hr and 18–20 hr AEL, respectively) and (C) one-way ANOVA (a, p>0.05 vs +/+ and b, p>0.05 vs +/+; *UAS-Hsl*).

Figure 6 with 1 supplement

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Differentiation of the embryo lipidome in control and *Hsl¹* mutants.

(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 glycerophospholipid classes of control and *Hsl¹* mutant embryos at different timepoints during embryogenesis (n = 8). (C) Relative changes of individual glycerophospholipid species in control and *Hsl¹* animals between early (0–2 hr AEL) and mid (6–8 hr AEL) or late (18–20 hr AEL) embryogenesis (n = 8). Data are presented as (**A, B**) means and SD or (C) log2-transformed fold changes (FC) normalized to early (0–2 hr AEL) control embryos.

Figure 6—figure supplement 1

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Changes in the lipidome during embryogenesis in control and *Hsl¹* mutants.

(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 AEL) and mid (6–8 hr AEL) or late (18–20 hr AEL) embryogenesis (n = 8). Data are presented as (**A, B**) means and SD or (C) log2-transformed fold changes (FC) normalized to early (0–2 hr AEL) control embryos.

Figure 7 with 1 supplement

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Impaired fecundity and egg loading in *Hsl¹* mutant flies.

(**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 lipids. (C) Lipid loading in control and *Hsl¹* mutant oocytes. Stage 10 follicles were stained with Hoechst 33342, BODIPY 493/503, and Cellmask Deep Red to detect nuclei, lipid droplets, and cell membranes, respectively, and imaged by confocal fluorescence microscopy. Scale bars: 50 µm. (D) Transfer of ³H-cholesterol into control and *Hsl¹* mutant embryos collected at different times after mating (n = 9–10). (**E, F**) Cumulative numbers of eggs and hatched 1st instar larvae per female upon (E) fat-body-specific RNAi-mediated downregulation of *Hsl* expression by means of a *Cg-GAL4-*driven *Hsl shRNA* transgene (n = 32–34) and (F) fat-body-specific rescue of *Hsl* expression in *Hsl¹* mutants by means of a *FB-GAL4-*driven *UAS-Hsl* transgene (n = 33–34). Data are presented as means and SD. Statistical difference was determined by (**A,B,D**) unpaired t-tests (#, p<0.05 vs control), (E) one-way ANOVA (a, c, p<0.05 vs Cg>+ at 4 days and 7 days after mating; b, d, p<0.05 vs *Hsl shRNA*/+ at 4 days and 7 days after mating) and (F) one-way ANOVA (a,b,c p>0.05 vs FB>+ at 2 days, 4 days, and 7 days after mating, respectively).

Figure 7—figure supplement 1

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Hatching rates of control and *Hsl¹* mutant embryos.

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 presented as means and SD. Statistical difference was determined by unpaired t-tests (#, p<0.05 vs control).

Tables

Key resources table

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)	Akh^A	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

Table 1

Drosophila strains used in the study.

Name in text/figure	Full genotype	ID/Reference
Control or +/+	w¹¹¹⁸; +; +	VDRC 60000
Hsl¹ or -/-	w¹¹¹⁸; Hsl¹; +	This study
Hsl^b24	w¹¹¹⁸; Hsl^b24; +	Bi et al., 2012
Akh^A	w¹¹¹⁸; +; Akh^A	Gáliková et al., 2015
bmm¹	w¹¹¹⁸; +; bmm¹	Gáliková et al., 2017
Akh^A bmm¹	w¹¹¹⁸; +; Akh^A bmm¹	This study
Hsl¹ bmm¹	w¹¹¹⁸; Hsl¹; bmm¹	This study
Hsl¹ Akh^A	w¹¹¹⁸; Hsl¹; Akh^A	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}Myo31DF^NP0001/Hsl¹; +*	This study, BDSC 67088
Hsl¹ Myo31DF UAS-Hsl	w; Hsl¹ P{w^+mW.hs=GawB}Myo31DF^NP0001/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}1*C/+	This study, BDSC 64277
-/-; nos-GAL4/UAS-Hsl	w; Hsl¹; P{w^+mC=GAL4::VP16-nos.UTR}1*C/ 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

Table 2

List of primers used for RT-qPCR.

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’