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Supplementary figure legends:
Fig. S1. Supplementary figure 1. (A) IGV plot exome sequencing from YUMM1.1,
YUMM1.7 and YUMM2.1 showing BRAFV600E mutation and PTEN exon 5
successfully removed by cre-mediated recombination (C57Bl/6 as normal comparator).
(B) Tumor growth curve of YUMM1.7 and B16 with 4 mice in each group (mean ± SD)
after anti-PD-1, anti-PD-L1 or isotype control. The arrow indicates the day when
treatment with anti-PD-1, anti-PD-L1 or isotype control was started. (C) Analysis of the
non-synonymous mutational load compared to a strain-matched normal with known
dbSNP variants excluded as assessed by the Enseml Variant Effect Predictor.
Fig. S2. Supplementary figure 2. (A) Quantification of CD3+CD8+ (CD8 T-cells) and
(B) CD3+CD4+ (CD4 T-cells) in both tumors and spleens in YUMM2.1. Tumor cells and
splenocytes were harvested on day 10 after starting treatment with anti-PD-1, anti-PD1+aCD8 (anti-PD-1aCD8), anti-PD-1+anti-CD4 (anti-PD-1aCD4), anti-PD-1 plus antiCD8 + anti-CD4 (anti-PD-1aCD8/4) or isotype control; with 4 mice in each group (mean
± SD). *P=0.004 anti-PD-1 versus control; P=0.003 anti-PD-1aCD8 versus control;
P=0.004 anti-PD-1aCD4 versus control; P=0.004 anti-PD-1aCD8/4 versus control, CD8
T-cells in tumors. *P<0.001 anti-PD-1 versus control; P=0.03 anti-PD-1aCD8 versus
control; P=0.03 anti-PD-1aCD4 versus control; P<0.001 anti-PD-1aCD8/4 versus
control, CD8 T-cells in spleens. *P<0.001 anti-PD-1aCD4 versus control; P=0.004 antiPD-1aCD8/4 versus control, CD4 T-cells in tumors. *P<0.001 anti-PD-1aCD4 versus
control; P<0.001 anti-PD-1aCD8/4 versus control, CD4 T-cells in spleens. (C) Gating
strategy after exclusion of dead cells of (D) CD3+CD8+ and CD3+CD4+ cells after gating
for CD3+ cells. Quantification of (E) CD3+CD8+ (CD8 T-cells) in MC38 and YUMM2.1
spleens. (F) CD3+CD4+ (CD4 T-cells) in MC38 and YUMM 2.1 spleens (G) CD8 T-cells
in both tumors and spleens in YUMM1.1. Tumor cells and splenocytes were harvested on
day 3 and 10 after starting treatment with anti-PD-1 or isotype control; with 4 mice in
each group (mean ± SD).
Fig. S3. Supplementary figure 3. (A) IGV plot of RNA-Seq from YUMM1.1,
YUMM1.7 and YUMM2.1. β-catenin exon 3 is included in all exon-junction spanning
reads, with no evidence of exon 2-4 junctions. (B) Western blot analysis of cytoplasmic
and nuclear β-catenin in YUMM1.7 and YUMM2.1 cell lines with or without exposure to
10 uM 4HT for 48 hours. (C) Top-flash activity of total β-catenin in YUMM1.7 and
YUMM2.1 with or without exposure to 10uM 4HT for 48 hours. (D) Representative
immunofluorescence of β-catenin stained non-treated tumors.
Fig. S4. Supplementary figure 4. (A) Gating strategy of CD11c+B220-, CD11c+B220+,
CD11c+B220-CD8+
and
CD11c+B220-CD103+
cells.
(B)
Gating
strategy
of
CD11b+MHC-IIhigh DCs in CD11c+ cells. (C) Gating strategy of CD11b+F4/80+TAMs,
CD11b+F4/80+MHC-IIlow TAMs and CD11b+F4/80+MHC-IIhigh TAMs. (D) Gating
strategy
of
MO-MDSC
(CD11b+Ly6ChighLy6Glow)
and
PMN-MDSC
(CD11b+Ly6ClowLy6Ghigh). (E) Gating strategy of Tregs (CD4+CD25+FoxP3+). (F)
Corresponding normalized enrichment scores (NES), P values and false discovery rates
(FDR) of the GSEA plots for YUMM2.1 versus YUMM1.1 enriched pathways involved
in immune response, cytokine production and inflammatory response.