Fig. 2

rt269L activates the PERK-mediated UPR pathway, but an impaired UPR is shown in rt269I-infected cells. A Markers of ER stress in Huh7, HepG2, and HepG2-NTCP-C4 cells, as measured by western blotting, after transfection or infection with mock, rt269L, or rt269I HBV. Some blots were cropped to improve the clarity and conciseness of the presentation. B Phosphorylation of eIF2α determined by immunofluorescence. HepG2 cells were transfected with a mock, rt269L, or rt269I vector and then immunostained with antibodies and DAPI and viewed by confocal microscopy. Scale bar, 10 µm. C Immunohistochemical analysis of p-eIF2α expression (red arrow) in paraffin-embedded liver tissues (magnification 100 ×, n = 5 per group). D, E ATF4 and HBV polymerase expression determined by immunofluorescence. F Left panel: agarose gel (3%) electrophoresis of XBP1 fragments (unspliced 295 bp, spliced 269 bp) from RNA isolation from HepG2 cells transfected with mock, rt269L, or rt269I HBV, or treated with thapsigargin (Tg, 1 µg/ml) for 6 and 12 h; right panel: RT–qPCR analysis of the transcription levels of UPR and ER stress-related genes 10 h posttransfection. Bar graph representing the relative expression levels (folds) of ATF4, ATF6, and sXBP1 calculated by the 2^−ΔΔCt method using β-actin as an endogenous control. **p < 0.01, ***p < 0.001