Figure 2. Transient low dose treatment of 5-AZA-CdR induces Type III Interferon production and activation of MAVS.

A) B16-Blue™ IFN-α/β Cells (InvivoGen) were transiently treated with 0.3uM of 5-AZA-CdR for 24 hours. Activity of the ISG54 promoter enhanced by a multimeric ISRE was determined by measuring secreted alkaline phosphatase (SEAP). SEAP activity was measured at 640 nm when the QUANTI-blue substrate was provided. The results presented are from three independent experiments. Error bars represent the SD of three independent experiments. B) Supernatants from B16 cells transiently treated with 5-AZA-CdR or transfected with 0.5ug/ml of poly(I:C) were added to wild-type B16-Blue™ IFN-α/β cells (InvivoGen), and the presence of bioactive IFNs-α/β were determined by measuring SEAP activity 24 hours after adding the supernatants. Error bars represent the SD of three independent experiments. C) B16-Blue™ IFN-α/β Cells (InvivoGen) were transiently treated with 5-AZA-CdR in the presence or absence of the JAK1/JAK2 inhibitor Ruxolitinib (1uM) or the JAK3 inhibitor CP-690550 (1uM). SEAP activity was measured at 640nm. D) LIM1215 colorectal cancer cell line was transiently treated with 5-AZA-CdR in the presence or absence of the JAK1/JAK2 inhibitor Ruxolitinib (1uM) or the JAK3 inhibitor CP-690550 (1uM). Gene expression of four selected interferon responsive genes was measured by real-time qPCR at day 5. E-F) LIM1215 with or without shRNA against MAVS were transiently treated with 5-AZA-CdR. Gene expression of the Type III interferon genes IL29 (E) and IL28a (F) was measured by real-time qPCR at day 5. G) Mitochondrial extracts were prepared from LIM1215 cells treated with 5-AZA-CdR for the indicated time, or transfected with Poly(I:C), and then aliquots of the extracts were analyzed by SDD-AGE. **** p<0.0001; ** p<0.01 (One-way ANOVA) See also Figure S2.