SUPPLEMENTARY MATERIAL
Archives of Toxicology
RNAi in murine hepatocytes - the agony of choice
-A study of the influence of lipid based transfection reagents in hepatocyte metabolism-
Jan Böttger, Katrin Arnold, Carlo Thiel, Christiane Rennert, Susanne Aleithe, Ute Hofmann, Sebastian Vlaic
Susanne Sales, Andrej Shevchenko, Madlen Matz-Soja*
*Corresponding author: Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Johannisallee 30,
04103 Leipzig, Germany
phone: +49 341 9722117, fax: +49 341 9722109, E-mail: madlen.matz@medizin.uni-leipzig
Table of contents
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1 Supplementary material and methods
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Shot-gun lipidomic analyses
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Fat red quantification
2 Supplementary tables
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Table S1: Oligos for RNA interference experiments
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Table S2: Primers for qRT-PCR analyses
3 Supplementary figures
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Figure S1: Time schedule for the experimental protocol
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Figure S2: Influence of transfection reagents on TAG species 24 h post transfection
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Figure S3: Influence of transfection reagents on TAG species 48 h post transfection
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Figure S4: Influence of transfection reagents on TAG species 72 h post transfection
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Figure S5: Influence of transfection reagents on lipidomic profile
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Figure S6: Hierarchical clustering of extracellular metabolite concentrations
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Figure S7: Influence of transfection reagents on metabolomic profile
Shotgun lipidomic analyses
Lipid extraction of mouse hepatocytes was performed using a modified Folch protocol (Folch et al., 1957). Briefly,
cells (an amount equivalent to ca 10 ug of total proteins) were dissolved in 200 µl of 150 mM ammonium
bicarbonate and 10 µl of an internal standard mixture containing: 20 pmol TAG 12:0-12:0-12:0, 20 pmol DAG 17:017:0, 40 pmol diethyl PC 18:0-18:0, 50 pmol diethyl PE 20:0-20-0, 10 pmol PG 17:0-17-0, 40 pmol PS 12:0-12:0,
50 pmol PI 16:0-16-0, 40 pmol LPC 12:0, 40 pmol LPE 14:0, 30 pmol SM d18:1-12:0, 90 pmol CE 12:0, 20 pmol
Cer d18:1-12:0, 50 pmol cholesterol d7 (all from Avanti Polar Lipids, Inc. Alabaster, AL, USA) were spiked in for
the subsequent quantification. Then, 265 µl of methanol and 730 µl of chloroform were added and the mixture
vortexed for 1 h at 4°C. The lower organic phase was collected, dried in a vacuum centrifuge and the lipid extracts
re-dissolved in 120 µl of a chloroform:methanol (1:2 (v/v)) mixture. The analysis was performed in negative and
positive ion mode. For negative mode analyses, 10 µl extract were mixed with either 12 µl of 13 mM ammonium
acetate in isopropanol or 0.1 % (v/v) triethylamine in methanol. For positive mode analyses, 10 µl extract were
mixed with 90 µl of 6.5 mM ammonium acetate in isopropanol before infusion. The analyses were performed on a Q
Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a robotic nanoflow ion
source TriVersa NanoMate (Advion BioSciences, Ithaca NY, USA). High resolution (140,000 at m/z 200) FT-MS
spectra were acquired for 1 min within the range of m/z 420-1000 in negative and 450-1000 in positive mode.
Cholesterol was quantified as previously described (Liebisch et al., 2006). Briefly, 30 µl of extract were dried under
vacuum, then 75 µl acetyl chloride:chloroform (1:2 (v/v)) were added, incubated for 1 h at RT, dried under vacuum
and re-dissolved in 60 µl chloroform:methanol (1:2 (v/v)). 10 µl extract were mixed with 90 µl of 6.5 mM
ammonium acetate in propanol before infusion and analyzed in positive mode. Lipid species were identified and
quantified using LipidXplorer software (Herzog et al., 2011).
Fat red quantification
Fat red quantification in nonsense-transfected hepatocytes was performed after fixation with 4 % paraformaldehyde
in PBS. Fixed cells were washed 3 times with distilled water followed by 60 % isopropanol for 10 min and were
subsequently tried. Afterwards cells were stained with Sudan red 7B (Serva, Heidelberg, Germany) for 10 min,
shortly treated with 60 % isopropanol and again washed 3 times with distilled water. After drying, 250 µl of 100 %
isopropanol were added to cells and incubated with constant shaking for 10 min. Absorption of 100 µl aliquots were
measured at 500 nm with a Spectra Max M5 (Molecular Devices, San Diego, USA). Values were normalized to
cellular protein.
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Table S1
Oligos for RNA interference experiments
Gene
Sequence
Ppar-
GACCGUCACGGAGCUCACAGAAUUU
AAAUUCUGUGAGCUCCGUGACGGUC
Ppar-
UCAAGGGUGCCAGUUUCGAUCCGUA
UCACGGAUCGAAACUGGCACCCUUGA
Apc
GGACUGGUAUUAUGCUCAATT
UUGAGCAUAAUACCAGUCCTT
Gata4
Mm_Gata4_3 FlexiTube siRNA (SI01009813)
Gli1
CCCAACAUGGCAGUGGGUAACAUGA
UCAUGUUACCCACUGCCAUGUUGGG
Gli2
CCACAACCACAACGUUGCUCAGACA
UGUCUGAGCAAGCUUGUGGUUGUGG
Gli3
UAGCAAGGCCAUCUUGGUCUUCAGG
CCUGAAGACCAAGAUGGCCUUGCUA
Smo
CGUAGCUUCCGGGACUAUGUGCUAU
AUAGCACAUAGUCCCGGAAGCUACG
Lxr-
CAGUGUCAUCAAGGGAGCACGCUAU
AUAGCGUGCUCCCUUGAUGACACUG
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Table S2
Primers for qRT-PCR analyses
Gene
Sequence
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Acox1
forward gcccaactgtgacttcca
reverse gccaggactatcgcatga
Actin-
forward catccgtaaagacctctatgccaac
reverse atggagccaccgatccaca
Apc
forward aggagaatgcagtcctgtcc
reverse ttgtgaggttctgaagttgagc
Cpt1a
forward gctgtcaaagataccgtgagc
reverse tctccctccttcatcagtgg
Gata4
forward ggaagacaccccaatctcg
reverse catggccccacaattgac
Gli1
Mm_Gli1_1_SG QuantiTect Primer Assay (QT00173537)
Gli2
Mm_Gli2_2_SG QuantiTect Primer Assay (QT01062236)
Gli3
Mm_Gli3_1_SG QuantiTect Primer Assay (QT00102256
Gys2
forward atcctttctcgtgccaggaa
reverse gcggtggtatatctgcctct
Fgf21
forward agatggagctctctatggatcg
reverse gggcttcagactggtacacat
Lxr-
forward aagggagcacgctatgtctg
reverse cttcttgccgcttcagtttc
Ppar-
forward cgtacggcaatggctttatc
reverse tcatctggatggttgctctg
Ppar-
forward atggaagaccactcgcattc
reverse gctttatccccacagactcg
Smo
forward gcaagctcgtgctctggt
reverse gggcatgtagacagcacaca
Figure S1
Figure S1: Time schedule for the experimental protocol
After hepatocyte isolation the cells were seeded in a 24-well plate. Towards 2 h of adhesion, the medium was
changed to serum-free medium (500 µl) which was used throughout further cultivation. Subsequently, the
hepatocytes were transfected with the siRNA or the nonsense oligo. A medium-change to normal culture medium
(500 µl) was performed 24 h and 48 h after transfection. At several time points cells were used for isolation of RNA
or collected for lipidomic and metabolomic profiling.
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Figure S2
Figure S2: Influence of transfection reagents on TAG species 24 h post transfection
Shot-gun lipidomic analyses of changes in Triacylglycerides species after 24 h in nonsense-transfected hepatocytes with INTERFERin® (n=3-6), Lipofectamine®RNAiMAX (n=3-6)
and HiPerFect® (n=3-6) as well as non-transfected control hepatocytes (control) (n=6). X-axis: The first number represents the chain length and the second number represents the
double bands. The significance was calculated to the control cultures for each time point, respectively.
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Figure S3
Figure S3: Influence of transfection reagents on TAG species 48 h post transfection
Shot-gun lipidomic analyses of changes in Triacylglycerides species after 48 h in nonsense-transfected hepatocytes with INTERFERin® (n=6), Lipofectamine®RNAiMAX (n=3-6)
and HiPerFect® (n=3-6) as well as non-transfected control hepatocytes (control) (n=3-6). X-axis: The first number represents the chain length and the second number represents the
double bands. The significance was calculated to the control cultures for each time point, respectively.
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Figure S4
Figure S4: Influence of transfection reagents on TAG species 72 h post transfection
Shot-gun lipidomic analyses of changes in Triacylglycerides species after 72 h in nonsense-transfected hepatocytes with INTERFERin® (n=6), Lipofectamine®RNAiMAX (n=3-6)
and HiPerFect® (n=3-6) as well as non-transfected control hepatocytes (control) (n=3-6). X-axis: The first number represents the chain length and the second number represents the
double bands. The significance was calculated to the control cultures for each time point, respectively.
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Figure S5
Fig. S5: Influence of transfection reagents on lipidomic profile
a-d: Shot-gun lipidomic analyses of changes in a: Sphingomyelines (SM), b: Lysophosphatitylcholine (LPC), c:
Lysophosphatidylethanolamine (LPE) and d: Ceramides (CER) levels in nonsense-transfected hepatocytes
transfected with INTERFERin® (n=6), HiPerFect® (n=6), Lipofectamine®RNAiMAX (n=6) as well as nontransfected hepatocytes (control) (n=6) 24 h, 48 h and 72 h post-transfection. Significance was calculated compared
with the control cultures for each time point, respectively.
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Figure S6
Figure S6: Hierarchical clustering of extracellular metabolite concentrations
Hierarchical clustering of the nonsense oligo-transfected groups (INTERFERin®, Lipofectamine®RNAiMAX and
HiPerFect® (n=7)) based on the metabolites with significant change in concentration over-time compared to the nontransfected group (control) (n=7) (see Fig. 5). Distance between the samples is reflected by height of the
dendrogram.
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Figure S7
Figure S7: Influence of transfection reagents on metabolomic profile
Concentration-time curve of the five significantly up- and down-regulated metabolites alanine, glycine, ornithine,
arginine and pyruvate, and the one non-significant regulated metabolite urea over the entire study period of 72 h
between the non-transfected group (control) (n=7) and the nonsense-transfected groups by using INTERFERin®,
Lipofectamine®RNAiMAX and HiPerFect® (n=7). Values are represented as means ± standard error (SD)
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