FIG 4.

GRP78-mediated inhibition of HBV replication was not due to the activation of innate immune responses. (A) HepAD38/Tet-off cells were transfected with increasing amounts of pGRP78 or a constitutively active mutant of RIG-I expression plasmid (pRIG-IN). At 48 h posttransfection, the cells were collected, and the level of IFN-β mRNA was tested by qRT-PCR. The data are means ± the SEM of five samples pooled from three independent experiments. (B) Cells were treated as in panel A. The mRNA level of MxA, ISG56, or TRIM22 was determined by qRT-PCR. The data are means ± the SEM of four samples pooled from three independent experiments. (C) Cells were treated as in panel A and then subjected to Western blotting with antibodies against p-TBK1, TBK1, p-IRF3, IRF3 GRP78, or GAPDH. (D) HepAD38/Tet-off cells were transfected with pcDNA or increasing amounts of pGRP78. At 48 h posttransfection, the caspase-1 activity was determined. The data are means ± the SEM of three samples pooled from three independent experiments. Cells stimulated with lipopolysaccharide (500 ng/ml) plus ATP (5 mM) were used as a positive control for inflammasome activation. (E) HepAD38 cells were treated as in panel D, and Western blotting was then performed with antibodies against caspase-1, IL-1β, GRP78, and GAPDH. (F) NF-κB promoter-dependent luciferase reporter plasmids (pNF-κB-Luc) were transfected into HepAD38 cells, together with pcDNA and increasing amounts of pGRP78 or pRIG-IN. After 48 h, the luciferase activity in the cell lysates was measured. The data are means ± the SEM of four samples pooled from three independent experiments. (G and H) Cells were transfected as in panel A. After 48 h, the mRNA level of TNF-α (G) or IL-6 (H) was determined by qRT-PCR. The data are means ± the SEM of four samples pooled from three independent experiments.