Characterization of stable BAC reporter cell lines.

(A) Schematic of the BAC reporter construct. Destabilization of the Mx1deGFP or IFIT1deGFP fusion protein was achieved by C-terminal fusion with the degradation domain of mouse ornithine decarboxylase (mODC). (B) A549-IFIT1deGFP cells were stimulated with 10 IU/ml IFN-α for 24 h and subjected to FACS to separate cells according to expression of IFIT1deGFP. Right after sorting, GFP-positive and -negative cells were lysed and total RNA was extracted. Amounts of mRNAs specified in the bottom of the graph were quantified by RT-qPCR and normalized to GAPDH mRNA levels. Note that the IFIT1 specific RT-qPCR detected both the endogenous ISG and the reporter mRNA. Data represent the mean from two independent experiments and their respective SDs. (C) A549-IFIT1deGFP cells were stimulated with 100 IU/ml IFN-α (to achieve high level expression of the endogenous ISG), harvested at time points specified in the top (hours) and analyzed by Western blot to detect proteins specified in the right. A representative blot from 3 independent experiments is shown. Mock-treated cells are shown in the right lane of each panel. (D) Induction kinetics of IFIT1deGFP and Mx1deGFP after stimulation of A549 reporter cell lines with IFN-α. Cells were treated with 100 IU/ml of IFN-α, harvested at time points specified in the bottom of the graph and number of GFP-positive cells was determined by flow cytometry. Data are mean from 3 independent experiments and their respective SDs. (E) IFN-α dose response assay with A549 reporter cell lines. Cells were stimulated for 24 h with 100 IU/ml IFN-α and analyzed for mean GFP intensity (left panel) or number of GFP-expressing cells (right panel) by using flow cytometry. Data are mean from 3 independent experiments and their respective SDs. (F) Half-life of IFIT1deGFP (left panel) and Mx1deGFP (right panel) as determined by CHX treatment of cells after pre-stimulation with 100 IU/ml IFN-α for 15 h. Cells were harvested at time points given in the bottom of the graph and cell lysates were analyzed by Western blot using GFP-, IFIT1-, Mx1- and β-actin-specific antisera. Representative blots are shown in the bottom of each panel; quantifications from 4 independent experiments and their respective SDs are depicted in the upper graphs of each panel. In panels C and F, numbers in the left of Western blots refer to molecular weights of size standards in kiloDalton (kDa), respectively.