Fig. S1 Efficient fibrosis induction after both carbon tetrachloride administration and bile duct
ligation. (a, b) Circulating alanine aminotransferase (a) and circulating aspartate aminotransferase (b)
were measured by a colorimetric activity assay in blood harvested at sacrifice from mice receiving Hepa16 cells expressing a GFP-luciferase vector implanted into C57Bl/6 background treated with (from left to
right) olive oil, carbon tetrachloride diluted in olive oil, sham surgery, or bile duct ligation. (c)
Representative picrosirius red staining for collage deposition in liver under the treatment conditions listed
above at low magnification (top row) and high magnification (bottom row). NS non-significant, **P<0.01,
****P<0.0001
Fig. S2 No differences in F4/80 expression in different Gr1 expression level subtypes of CD11b+
monocytes. (a, b, c) Quantification of mean fluorescence intensity of F4/80 staining on tumor-infiltrating
CD11b+Gr1hi (a), CD11b+Gr1mid (b), and CD11b+Gr1lo (c) cells in C57Bl/6 mice treated with (from left to
right) olive oil, carbon tetrachloride diluted in olive oil, sham surgery, or bile duct ligation. NS nonsignificant
Fig. S3 Alterations in the lymphocytic and myeloid immune contexture of fibrotic tissue in the CCl4
model of fibrosis induction. (a) Quantification of Singlets (live single cells based on FSC-A and FSC-H
analyses following SSC-A and FSC-A gating), NK1.1+, NK1.1+TCR+, TCR+, TCR+CD4+,
TCR+CD8+, and TCR+CD4+CD25+ cells in non-tumor liver tissue from mice treated with olive oil or
CCl4 as percentage of total recorded events. (b) Quantification of NK1.1+, NK1.1+TCR+, TCR+,
TCR+CD4+, TCR+CD8+, and TCR+CD4+CD25+ cells in non-tumor liver tissue from mice treated with
olive oil or CCl4 as percentage of gate. (c) Quantification of Singlets (live single cells based on FSC-A
and FSC-H analyses following SSC-A and FSC-A gating), CD11b+, CD11b+Gr1hi, CD11b+Gr1mid, and
CD11b+Gr1lo cells in non-tumor liver tissue from mice treated with olive oil or CCl4 as percentage of total
recorded events. (d) Quantification of CD11b+, CD11b+Gr1hi, CD11b+Gr1mid, and CD11b+Gr1lo cells in
non-tumor liver tissue from mice treated with olive oil or CCl4 as percentage of gate. (e) Quantification of
mean F4/80 fluorescence intensity of CD11b+, CD11b+Gr1hi, CD11b+Gr1mid, and CD11b+Gr1lo cells in
non-tumor liver tissue from mice treated with olive oil or CCl4. *P<0.05, **P<0.01, ***P<0.001
Fig. S4 Clear separation of GFP+ tumor cells and GFP- non-tumor cells. Tumors were harvested at 14
days post-implantation, and a single cell suspension was made. Tumor cells expressing a GFP-luciferase
vector were sorted from the suspension. Shown is a representative separation, with clear distinction
between viable GFP+ and GFP- cells
Fig. S5 Soluble immune mediator expression by tumor-infiltrating stromal cells developed in a
fibrotic liver is markedly different from that in a healthy liver. Tumors were harvested at 14 days
1
post-implantation, and a single cell suspension was made. Tumor-infiltrating stromal cells negative for
GFP were sorted from the suspension, their RNA extracted and reverse transcribed, and expression of
soluble immune mediators examined. Comparison of expression of cytokines in stromal cells isolated
from tumors implanted into either olive oil- (red) or carbon tetrachloride- (blue) treated mice, normalized
to average expression of these factors in tumor-infiltrating stromal cells implanted into mice which were
treated with olive oil.
Table S1 Expression of cytokines in tumor-infiltrating stromal cells isolated from tumors implanted
into olive oil- or CCl4-treated mice relative to average level of expression in the olive oil-treated mice.
This table contains both summary for treatment groups and raw data for each mouse used in the analysis.
Fold changes were generated using the formula:
Fold Change = 2^-(sample Ct value – average of olive oil Ct values)
Table S2 Expression of cytokines in tumor cells implanted into olive oil- or CCl4-treated mice
relative to expression in the same cell line maintained in culture in vitro. This table contains both
summary for treatment groups and raw data for each mouse used in the analysis. Fold changes were
generated using the formula:
Fold Change = 2^-(sample Ct value – in vitro Ct value)
Table S3 Expression of cytokines in tumor cells implanted into olive oil- or CCl4-treated mice
relative to average expression in the olive oil-treated mice. This table contains both summary for
treatment groups and raw data for each mouse used in the analysis. Fold changes were generated using the
formula:
Fold Change = 2^-(sample Ct value – average of olive oil Ct values)
2
Fig. S1:
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Fig. S5:
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