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. Author manuscript; available in PMC: 2022 Jun 16.
Published in final edited form as: J Immunol. 2017 Jul 31;199(5):1846–1855. doi: 10.4049/jimmunol.1601493

FIGURE 3. PACT augments dsRNA-induced activation of MDA5.

FIGURE 3.

(A) HEK293 cells carrying a stably expressed IFN-β–luc plasmid were transfected with PACT and MDA5 expression plasmids for 24 h before poly(I:C) induction (1 μg/ml). A luciferase reporter assay was carried out; relative luciferase activity is shown in arbitrary units. Data are the mean and SD of three replicates. *p < 0.05, PACT + MDA5 versus PACT at the same time point, Student t test. WT and PACT−/− MEFs were induced with poly(I:C) as above and analyzed for the expression of Ifnb (B) and Ifna4 (C) mRNAs. *p < 0.05, PACT−/− versus WT at the same time point, Student t test. (D) PACT was reintroduced into PACT−/− MEFs by ectopic expression 24 h prior to poly(I:C) induction at 1 μg/ml. The fold induction of the Ifnb gene was calculated with respect to GAPDH transcripts in mock-treated cells at time zero. *p < 0.05, PACT versus vector at the same time point, Student t test. (E) HEK293 cells were rendered deficient in PACT expression by double-nicking via CRISPR/Cas9-mediated genome editing. Two positive clones (clones 1 and 2) were validated for the expression of PACT protein by Western blotting using parental untreated WT cells as control (left panel). Luciferase reporter assay of IFN-β promoter activity was performed in these cells with overexpression of MDA5 and induction by poly(I:C) (right panel). Results are representative of three independent experiments. Error bars indicate SD. *p < 0.05, clones 1/2 versus WT, Student t test. ND, not detected.