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Supplemental Figure 1. Loss of Stat3 expression did not change IFN-induced chemokine levels in tumors. (A) Realtime PCR analysis of mRNA expression levels of CXCL9, CXCL10, CXCL11 and (B) ELISA of CXCL10 levels in tumor single-cell suspensions from B16 tumor bearing Stat3 +/+ (Stat3 f/lf ) or Stat3 -/- (CD4Cre/Stat3 f/f ) mice. A B ns
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Stat3 Stat3 *** A B Supplemental Figure 2. Tumor infiltrating macrophages are expressing CXCL10. (A) Left, representative flow plot of sorted macrophages gated on CD11b + cells, (B) Real-time PCR analysis of CXCL10 mRNA levels in CD11b + F4/80 + macrophages, from B16 tumors harvested from Stat3 / or Stat3 / mice. F4/80 CD11b Stat3 Stat3 80.6% 79.0%
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CXCR3 CD8 Stat3 Stat3 24.3% 24.4% ns Supplemental Figure 3. Ablating Stat3 resulted no significant change in CXCR3 levels in CD8 + T cells from TDLN. Left, representative flow plot of cells from TDLN in B16 tumor bearing Stat3 +/+ or Stat3 -/- mice, gated on CD3 + CD8 +. Right, quantification analysis of percentages of CXCR3 + in CD3 + CD8 + T cells. Ns, not significant.
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ns * Supplemental Figure 4. Blockade of IFNγ in tumors lead to significant reduction of tumor infiltrating IFNγ-expression CD8 + T cells in absence of Stat3 expression. Flowcytometry analysis of IFNγ- expressing tumor infiltrating T cells (first gated on CD3 + CD8 + ) from B16 tumor bearing Stat3 +/+ or Stat3 -/- mice. Quantification of CD8 + IFNγ + percentages are showing on the right. PBS αIFNγ 4.39% 8.37% 3.10% 2.29% IFNγ CD8 Stat3 Stat3 PBSαIFNγ
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Supplemental Figure 5. CXCL10 induced CD8 + T cell migration depends on Rho signaling but independent of Cdc42 and Rac1 signaling. (A)-(B) In vitro migration of splenic CD8 + T cells from B16-bearing Stat3 +/+ or Stat3 -/- mice toward medium alone or CXCL10 at 100ng/ml. Cells were pretreated with ROCKi (Rho kinase inhibitor, 10 μM for 15 min), CT04 (Rho A family inhibitor, 1 μg/ml for 2hrs), ML141 (Cdc42 inhibitor, 10 μM for 1hr) and NSC23766 (Rac1 inhibitor, 100 μM for 1hr) before migration assay. ** A B ns
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IFNγ Stat3 IFNγ CXCL10 CD8 + T cell Myeloid cell Supplemental Figure 6. Illustration of STAT3 signaling in regulating CD8 + T cells migration toward tumors in an IFNγ/CXCL10/CXCR3- dependent manner.
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