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. 2018 Oct 29;92(22):e00825-18. doi: 10.1128/JVI.00825-18

FIG 2.

FIG 2

PJA1 generally represses viral and episomal DNAs independent of IFN signaling pathways. (A and B) 293T cells (A) or HepG2 cells (B) were plated in 12-well plates and transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h. The mRNA levels of endogenous IFN-α, IFN-β, and IFN-γ genes were measured by RT-qPCR. (C to E) 293T cells were plated in 12-well plates; transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h; and treated with recombinant human IFN-α (rhIFN-α) (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The mRNA levels of endogenous PKR (C), OAS1 (D), and MX1 (E) genes were measured by RT-qPCR. (F) HepG2 cells were plated in 12-well plates; transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h; and treated with rhIFN-α (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The mRNA level of the endogenous PKR gene was measured by RT-qPCR. (G and H) 293T cells (G) or HepG2 cells (H) were treated with rhIFN-α (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The PJA1 and GAPDH mRNA levels were measured by RT-qPCR. (I) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA or pCAGGS-HA-PJA1B and 0.2 μg reporters, pIFN-β-Luc, pISRE-Luc, pNF-κB-Luc, pTp53-Luc, and pCMV-Luc for 24 h. Luciferase activities were measured. (J) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1B and 0.2 μg pCMV-Luc for 24 h. Luciferase activities were measured. (K to M) 293T cells were plated in 24-well plates and cotransfected with 0.2 μg pCMV-Luc and pCAGGS-HA-PJA1B at 0, 0.01, 0.05, and 0.1 μg for 24 h. (K) Luciferase activity was measured. (L) The Luc mRNA level was quantified by RT-qPCR. (M) pCMV-Luc DNA in the nuclear extract was quantified by qPCR. (N to P) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA, pCAGGS-HA-PJA1B, or pCAGGS-HA-PJA1BΔR and 0.2 μg reporters, pISRE-Luc (L), pCMV-Luc (M), and pTK-Renilla-Luc (N) for 24 h. Luciferase activities were measured. (Q and R) 293T cells were plated in 12-well plates and transfected with 0.5 μg pEGFP and 0.5 μg pCAGGS-HA, pCAGGS-HA-PJA1B, or pCAGGS-HA-PJA1BΔR for 24 h. (Q) GFP, PJA1, and β-actin were detected by Western blotting. WCL, whole-cell lysate. (R) Fluorescence intensity of EGFP was detected. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. Results are expressed as fold induction relative to the control. ns, not significant (P > 0.05); *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001.