Supplementary Figure Legends
Supplementary Figure 1 (a) Macroscopic appearance of 4 representative WT,
NemoΔhepa, NemoΔhepa/TRAIL-/- and NemoΔhepa/TNFR1-/- livers at 8 weeks of age.
Arrows indicate widespread lesions. (b) Macroscopic nodules were quantified in
NemoΔhepa, NemoΔhepa/TRAIL-/- and NemoΔhepa/TNFR1-/- mice at 8 weeks of age and
represented n=26, p<0.05). (c) Representative Oil Red-O staining in liver sections of
8 week-old WT, NemoΔhepa, NemoΔhepa/TRAIL-/- and NemoΔhepa/TNFR1-/- animals
(n=4, p<0.01-0.001). (d) 500.000 primary hepatocytes from 8 week-old WT,
NemoΔhepa and NemoΔhepa/TNFR1-/- were isolated and cultured. Arrows indicate dead
cells. (e) MTT assay for hepatocyte survival after 8 h incubation (n=4, ***p<0.010.001).
Supplementary Figure 2 (a) Hepatocyte proliferation was analyzed in 8 week-old
animals by Ki-67 staining (red). (b) BrdU-positive cells per high magnification fields
(n=4; 5 fields per animal; *p<0.05; **p<0.01) in livers of 8 weeks-old WT, NemoΔhepa,
NemoΔhepa/TRAIL-/- and NemoΔhepa/TNFR1-/- animals. RT-PCR quantification of Cyclin
A2 (c), mRNA fold induction of p21 (d), mRNA relative expression of Collagen IA1 (e)
and α-SMA (f). Results are expressed as mean ± SEM (n=4, *p<0.05).
Supplementary Figure 3 (a) Percentage of liver infiltrating Ly6G after FACS
analysis. (b) CD11b cells-positive cells were detected
and quantified by
immunofluorescence of liver cryosections of WT, Nemo Δhepa, NemoΔhepa/TRAIL-/- and
NemoΔhepa/TNFR1-/- animals. (c) Representative pictures and quantification of Ly6Gpositive cells in livers of 8 weeks-old WT, NemoΔhepa, NemoΔhepa/TRAIL-/- and
NemoΔhepa/TNFR1-/- animals. Results are expressed as mean ± SEM (n=4, **p<0.01;
***p<0.001).
(d)
Liver
protein
extracts
from
8
week-old WT,
NemoΔhepa,
NemoΔhepa/TRAIL-/- and NemoΔhepa/TNFR1-/- were subjected to immunoblotting using
antibodies against TNFR1 and β-actin as loading control.
Supplementary Figure 4 The profile expression of genes related to liver fibrosis,
hepatic stellate cell activation, acute phase response, death receptor signalling and
hepatic
cholestasis
were
plotted
using
the
Venny
software
(http://bioinfogp.cnb.csic.es/tools/venny/index.html). Livers of 8 weeks-old liver tissue
from WT, NemoΔhepa, NemoΔhepa/TRAIL-/-, NemoΔhepa/TNFR1-/- and NemoΔhepa/TRAIL-//TNFR1-/- were used (n=3 livers per group). Representative diagram of the overlap of
top up-regulated (a) or down-regulated (b) genes normalized to WT mice.
Quantitative RT-PCR analysis of the mRNA levels of DR6 (c) and SCD2 (d),
respectively, in livers of 8 week-old WT, NemoΔhepa, NemoΔhepa/TRAIL-/- and
NemoΔhepa/TNFR1-/-. Values are mean ± SEM from at least 3 mice per group
(*p<0.05).
Supplementary Figure 5 Nodules greater than 5 mm were quantified in livers of
TRAIL-/-/TNFR1-/-,
NemoΔhepa,
NemoΔhepa/TRAIL-/-,
NemoΔhepa/TNFR1-/-
and
NemoΔhepa/TRAIL-/-/TNFR1-/- mice at the age of 52 weeks.
Supplementary Figure 6 (a) 4-6 weeks-old NemoΔhepa mice were reconstituted with
bone marrow from WT and TNFR1-/- mice. 52-weeks after transplantation mice were
sacrificed and liver samples collected. Macroscopic appearance of livers and
representative H/E staining is shown at 20x magnification. (b) Serum AP levels of
bone marrow transplanted mice were determined 1 year after transplantation. (c)
Nodules greater than 5 mm were quantified in livers of mice, 52 weeks after BMT.
Results are expressed as mean; error bars indicate SEM (n=6-8, p<0.01-0.001).