Fig. 3: Generation of stable cell lines expressing hNTCP, pNTCP, and phNTCP variants (157-167) and (S158G; P167L).
From: Identification of amino acids restricting HBV receptor function in porcine NTCP
HepG2 cell lines stably expressing hNTCP, pNTCP, phNTCP B1 (157-167), and phNTCP B5 (S158G; P167L) were generated. A Cells were treated with fluorescently labeled MyrB565 and analyzed via fluorescence microscopy to investigate HBV binding (scale bar: 100 µm). B Cells were seeded with low density and treated with Atto488-labeled MyrB (MyrB488). Membrane structure was visualized with Alexa Fluor647 labeled wheat germ agglutinin (WGA647), and nuclei were stained with Hoechst33342. High-resolution confocal microscopy was performed to investigate the correct location of the NTCP variants expressed (scale bar: 10 µm). In order to visualize the colocalization of WGA647 and MyrB488, fluorescence intensities along white indicators were compared. C Cells were seeded, differentiated, and inoculated with HBV (MOI 500 vp/cell). Supernatants were collected and analyzed for HBeAg at days 4 and 7 post-infection. The dotted line indicates the cut-off between non-reactive and reactive. Experiments were performed in biological triplicates; mean values +/− standard deviation are given. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison correction. *p < 0.05, **p < 0.01, ****p < 0.0001.
