Pro-apoptotic Sorafenib-signaling in murine hepatocytes depends on malignancy and is associated with PUMA expression in vitro and in vivo

Roland Sonntag, Nikolaus Gassler, Jörg-Martin Bangen, Christian Trautwein

and Christian Liedtke

Supplementary Material and Methods

Supplementary Table 1: Primer sequences used for quantitative real-time PCR

gene / annealing temperature / orientation / sequence in 5´-3´orientation
Bcl-Xl / 60°C / sense / GCT GCA TTG TTC CCG TAG AG
antisense / GTT GGA TGG CCA CCT ATC TG
Bcl-2 / 60°C / sense / GCT GAG CAG GGT CTT CAG AG
antisense / AGT ACC TGA ACC GGC ATC TG
Cyclin A2 / 59°C / sense / GTG GTG ATT CAA AAC TGC CA
antisense / AGA GTG TGA AGA TGC CCT GG
Cyclin D1 / 60°C / sense / AAG CAT GCA CAG ACC TTT GTG G
antisense / TTC AGG CCT TGC ATC GCA GC
Cyclin E1 / 59°C / sense / TCC ACG CAT GCT GAA TTA TC
antisense / TTG CAA GAC CCA GAT GAA GA
GAPDH / 58°C / sense / TGT TGA AGT CAC AGG AGA CAA CCT
antisense / AAC CTG CCA AGT ATG ATG ACA TCA
Mcl-1 / 59°C / sense / TAA ATA ACC CCT CCC CCA TC
antisense / AAG CAT GCC AAT CCA AGA AT
p21 / 58°C / sense / TTG CAC TCT GGT GTC TGA GC
antisense / TCT GCG CTT GGA GTG ATA GA
PUMA / 58°C / sense / ACC TCA ACG CGC AGT ACG
antisense / GGG AGG AGT CCC ATG AAG AG

Supplementary Table 2: Antibodies used for immunoblot analysis and in situ staining

Primary Antibodies (detail information) / Company
Akt / Cell Signaling
Bcl-2 (N-19) / Santa Cruz Biotechnology, Inc
Bcl-Xl (H-62) / Santa Cruz Biotechnology, Inc
BrdU (RPN202) / GE Healthcare
Cleaved Caspase-3 (Asp175) / Cell Signaling
Cyclin A2 (C-19) / Santa Cruz Biotechnology, Inc.
Cyclin B1 (M-20) / Santa Cruz Biotechnology, Inc.
Cyclin D1 (72-13G) / Santa Cruz Biotechnology, Inc.
Cyclin E (M-20) / Santa Cruz Biotechnology, Inc
GAPDH (MCA4739) / Abd Serotec
Ki-67 (NCL-Ki67p) / Leica Microsystems
Mcl-1 (600-401-394) / Rockland
P21 (SXM30) / BD Pharmingen
PCNA (PC10) / Invitrogen
p44/42 MAPK (Erk1/2) (137F5) / Cell Signaling
Phospho-Akt (Ser473) (193H12) / Cell Signaling
Phospho-Mdm2 (Ser166) / Cell Signaling
Phospho-MEK1/2 (Ser217/221) (41G9) / Cell Signaling
Phospho-p44/42 (Erk1/2) (Thr202/Tyr204) (197G2) / Cell Signaling
Phospho-Rb (Ser807/811) / Cell Signaling
Puma (Rodent Specific) / Cell Signaling
ß-Actin (A2066) / Sigma Aldrich
Secondary Antibodies / Company
Chicken anti-mouse IgG-HRP / Santa Cruz Biotechnology, Inc.
Goat anti-rabbit IgG-HRP / Santa Cruz Biotechnology, Inc.
Alexa 488 goat anti-mouse / Invitrogen
Alexa 546 goat anti-rabbit / Invitrogen

Legends to Supplementary Figures

Supplementary Fig. 1.Sorafenib-mediated signaling in Hepa1-6 cells and primary hepatocytes. (A, related to Fig. 3B) Primary hepatocytes were mitogen-stimulated with EGF and insulin. Left: Kinetics of Cyclin A2 mRNA expression in mitogen-stimulated hepatocytes without Sorafenib treatment is shown demonstrating maximal induction 48–72 h after plating. Right: Sorafenib-mediated dose-dependent reduction of Cyclin A2 gene expression measured 72 h after plating. (B, related to Fig. 3F) Primary hepatocytes were treated with increasing concentrations of Sorafenib as indicated and co-stimulated with EGF and insulin. Cells were analysed for BrdU incorporation by fluorescence microscopy after 72 h of mitogen stimulation. BrdU-positive nuclei are stained in green (arrows); total nuclei are counter-stained with DAPI (blue). Controls (0 µM, left) did not receive mitogens.(C, related to Fig. 4A) Primary hepatocytes were treated with increasing concentrations of Sorafenib, the solvent DMSO (0m) or left untreated (w/o) and analyzed for Mcl-1 protein expression. Equal loading was confirmed by determination of -Actin expression.

Supplementary Fig. 2. Sorafenib triggers transient cell cycle inhibition and increased liver injury after PH.WT mice received Sorafenib or vehicle 2 hours before PH and every 24h or 6h before sacrificed. Controls were either left completely untreated (0h ctrl) or subjected to PH only (48h ctrl).(A)Western blot analysis of cell-cycle related proteins after PH. Data is related to Fig. 5F showing a second, independent experiment.(B) Liver paraffin sections from mice treated with Sorafenib (Sora) or vehicle (vec) 96h after PH and were stained with Chloroacetate Esterase (CAE) to identify granulocytes. CAE positive granulocytes (red) are highlighted by arrows. (C) Quantitative analysis of CAE positive granulocytes perHPF. HPF: 200X magnification field.*: p<0.05; **: p<0.01; ***: p<0.001; n.s.: not significant.

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