Supplementary Figure 3: EGFR is required for TEX-mediated innate antiviral immunosuppression
From: Tumor-derived exosomes antagonize innate antiviral immunity
(a) Immunoblot analysis of total cell lysate (TCL) and exosomes (Exo) derived from parental and EGFR-deficient LLC cells. (b) qPCR (left, middle) of Ifnb1 mRNA expression and ELISA analysis (right) of IFN-β secretion in primary peritoneal macrophages pretreated with exosomes (40 μg) derived from MLF (control, Co.), LLC (EGFR+), and EGFR-deficient LLC (EGFR−) cells for 24 h, followed by stimulation with SeV (left) or transfection with poly (I:C) (middle) for 8 h. (c) qPCR analysis of Ifnb1 mRNA (left) and VSV specific mRNA (middle), and plaque assay of VSV titer (right) in primary peritoneal macrophages pretreated with exosomes (40 μg) derived from MLF (control, Co.), LLC (EGFR+), and EGFR-deficient LLC (EGFR−) cells for 24 h, followed by treatment with PBS or VSV (MOI,0.1) for 8 h. (d) Left panel: fluorescence microscopy of VSV–GFP amplification in HEK293T cells pretreated with exosomes (40 μg) derived from MLF (Co.), LLC and EGFR-deficient LLC cells for 24 h, followed by infection for 18 h with VSV–GFP (MOI 0.1) (bright-field, upper; fluorescence, bottom). Scale bars, 100 μm. Right panel: VSV-GFP intensity shown in the left panel was quantified by Image J. (e) qPCR analysis of Ifnb1 mRNA (left) and HSV-1 replication (middle), and plaque assay of HSV-1 titer in primary peritoneal macrophage cells pretreated with exosomes (40 μg) derived from MLF (control, Co.), LLC (EGFR+), and EGFR-deficient LLC (EGFR−) cells for 24 h, followed by treatment with PBS or HSV-1 (MOI,10) for 8 h. (f) qPCR of VSV mRNA in the lung (left), spleen (middle) and liver (right) of mice treated as in Fig. 2g. (g) Plaque assay of VSV titers in the lung (left), spleen (middle) and liver (right) of mice treated as in Fig. 2g. (h) qPCR analysis of HSV-1 gDNA (left) and plaque assay of HSV-1 titers (right) in the brains of mice treated as in Fig. 2g. All qPCR and plaque assay results are shown as mean + s.d. or mean ± s.d. of triplicates of at least two independent experiments. *P < 0.05.
