Fig 1: Reduced temperature also affects IFN-α/β induction. (A to C) MEFs were transfected with 50 µg poly(I:C) per million cells using electroporation. Pooled transfections were then divided among 30, 37, and 39°C temperature conditions. (A) Supernatants were assayed for biologically active IFN-α/β as described in Materials and Methods. Data are presented as log10 mean IU per milliliter ± SD. (B) qRT-PCR was used to quantify IFN-α/β and IFIT1 mRNA induction. Data are presented as mean fold change in 18S rRNA-normalized CT values in poly(I:C)-treated versus untreated cells at each temperature ± SD. (C) Immunoblot assay of IRF3 phosphorylated at Ser-396 and IFIT1 from poly(I:C)-treated MEF lysates. (D to F) Supernatants from virus-infected MEFs or RAW 264.7 cells were assayed for IFN-α/β as in panel A. IFN-α/β induction data are presented on the left y axis as log2 mean IU per milliliter ± SD. Statistics: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant by two-way ANOVA with Tukey’s multiple-comparison test of log-transformed IU/milliliter values. Viral titer data for panels D and E were determined by plaque assay on BHK cells at 37°C from the same supernatants assayed for IFN-α/β activity and are presented along the right y axis of those panels, ± SD. #, result was below the limit of detection (LOD) of the assay. All experiments were done in duplicate or triplicate, and results shown are representative of at least two independent experiments.