Figure 5.

Knockout of NFATC3 reduces CpG DNA–induced IFN-α and inhibits IRF7 nuclear translocation, and NFATC3 restoration rescues the responses. (A–C) CRISPR knockout Gen2.2 cells were stimulated with different doses of CpG A. NT guide RNA–transduced cells were used as controls. Production of IFN-α, TNF-α, and IL-6 were measured by ELISA. P-values are NT compared with NFATC3 knockout Gen2.2 cells. (D) CRISPR knockout Gen2.2 cells were stimulated with CpG A for 3 h. IRF7 and p50 nuclear translocation were analyzed by immunoblotting. Anti-transferrin receptor (Tfr) antibody and anti-HDAC1 antibody were used to show the cytoplasmic fraction and nuclear fraction. (E) NFATC3 knockout Gen2.2 cells that were stably transduced with mutated NFATC3 expression lentiviral particles that contained the mutated guide RNA–targeting sequence (NFATC3a) to restore NFATC3 expression. The lentiviral particles that contained empty lentiviral vector were used as a control. NFATC3 expression level was measured by immunoblotting. (F) NFATC3 knockout and NFATC3-restored Gen2.2 cells were stimulated with CpG A for 24 h. Production of IFN-α was measured by ELISA. Data are presented as the mean ± SEM of triplicate wells and are representative of three independent experiments. *, P < 0.05; **, P < 0.01 by Student’s t test.