Fig.S1 Efficient Fibrosis Induction After Both Carbon Tetrachloride Administration And

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 Hepa1-6 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 non-significant

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+Gr1locells 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+Gr1locells 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.S5Soluble 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 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 S2Expression of cytokines in tumor cells implanted into olive oil- or CCl4-treated mice relative to expression in the same cell line maintained in culturein 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)

Fig.S1:

Fig.S2:

Fig.S3:

Fig.S4:

Fig.S5: