Fig. 5

rt269I induces caspase activation and cell death by releasing cytochrome c. A Confocal images of HepG2 cells transfected with the rt269L or rt269I vector. Green fluorescence represents active (cleaved) caspase-3. Mitochondrial features were stained with MitoTracker, and nuclei were labeled with DAPI. Scale bar, 5 µm. B Western blot analysis of cleaved caspase-3 in HepG2 cells transfected with the rt269L or rt269I vector. The relative intensity of cleaved caspase-3 is compared with GAPDH. ***p < 0.001. C Immunohistochemistry analysis of cleaved caspase-3 expression in paraffin-embedded liver tissues (magnification 100 ×, n = 5 per group). D Left panel shows detection of cytochrome c in liver tissues of mice hydrodynamically injected with mock, rt269L, or rt269I vector and determined by ELISA. Right panel shows cytotoxicity levels measured in mock-, rt269L-, or rt269I-type HBV-infected HepaRG cells. E Cell death in liver tissues of mice hydrodynamically injected with mock, rt269L, or rt269I vector was detected by TUNEL assay (FITC-conjugated). F Detection of apoptotic HepG2 cells using Annexin V-FITC and 7-aminoactinomycin D (7-AAD) double-positive assays. Staurosporine (1 µM) was used to detect the positive control (Con), and the percentage of early or late apoptotic cells is indicated. G Mouse liver sections stained with H&E to analyze the liver injury and necrosis area among mouse groups hydrodynamically injected with mock, rt269L, or rt269I HBV. Magnification, 100×