Supplementary Methods
Immunohistochemistry and Western Blotting
Livers were fixed in 10% formalin overnight, embedded in paraffin and stained with H&E for
routine histopathological examination. For Oil Red O staining, frozen liver tissues were cut at
5 µm, mounted on slides and immediately stained and embedded according to
manufacturer’s instructions (Certistain (Merck), selected tissues were counterstained with
haematoxylin. Whole cell lysates were prepared from frozen cell pellets using RIPA Buffer
(50 mM TRIS pH 7,4, 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 1 % Na-deoxycholat,
0,1 % SDS) containing Complete Protease Inhibitor Cocktail (Roche) and sonication.
Proteins were separated by SDS-PAGE and transferred onto Immobilon-P PVDF (Millipore).
Membranes were probed with specific antibodies against alpha-fetoprotein (R&D Systems,
AF5369), Igf-2 (abcam, ab9574), SirT6 (Cell Signaling Technology, #2590) and -tubulin
(Sigma-Aldrich, T 7816). Immune complexes were detected using CDP-Star (Applied
Biosystems) as substrate for alkaline phosphatase-coupled secondary antibodies (SigmaAldrich).
Quantitative Real-Time Reverse-Transcriptase Polymerase Chain Reaction
Differential expression of the selected genes was measured using quantitative real-time
reverse-transcriptase polymerase chain reaction (qRT-PCR). A two-step qRT-PCR, using the
iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories), ABsoluteTM Blue QPCR SYBR®
Green Mix (Thermo Scientific) and the Stratagene Mx3000PTM QPCR system was
performed. Oligonucleotide primer sequences are listed in Supplemental Table 4. RPII was
used as a reference. The relative expression level of each gene was calculated using the
formula 2(-ΔΔCt).
MicroRNA quantification by qRT-PCR
Small RNAs (>18 nt) were isolated using the miRNeasy Mini Kit (Qiagen). The miScript PCR
Starter Kit (Qiagen) was used for reverse transcription and qRT-PCR quantification of
miRNAs.
The
miRNA
675-5p
was
quantified
with
the
oligonucleotide
primer
GTGCGGAAAGGGCCCACAG. A primer for quantification of the snRNA RNU6B contained
in this kit served as a control for normalization of qPCR results. A universal reverse primer
for qRT-PCR was also provided.
Transfection and viability assays
Transfections were carried out with Transfectin Lipid Reagent (Bio-Rad) according to the
manufacturer’s protocol. Cells were transfected in petri dishes and then re-plated to 96-well
plates for cell viability assays. The Sirt6 expression vector was pCMV6-Entry-Sirt6 (OriGene,
NM016539.1) HepG2 cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM).
Cells were treated in 4 replicates with 1 µg/mL and 10 µg/mL Doxorubicin or Daunorubicin
(provided by the pharmacy of the University Medical Center, Mainz) or 100 ng/mL anti-Apo-1
for 24 h. CD95 agonistic antibody (anti-APO-1) was a generous gift from Peter Krammer
(DKFZ, Heidelberg). Cell viability was measured with the CellTiter-Glo® Luminescent Cell
Viability Assay (Promega) according to the manufacturer’s protocol.