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Fig. 3 | BMC Medicine

Fig. 3

From: Occult infection with hepatitis B virus PreS variants synergistically promotes hepatocellular carcinoma development in a high-fat diet context by generating abnormal ceramides

Fig. 3

Infections with PreS variants induced hepatocyte ER stress and augmented the generation of abnormal ceramides. HepG2 cells were transfected respectively with different HBV plasmids as described in Fig. 2D and cultured for 48 h. Empty-vector (Vec) was the transfection control. A Transcriptional levels (mean ± SEM) of the specified genes from three independent experiments determined by qRT-PCR. The other genes are provided in Additional file 1: Fig. S6. Image shows one representative of 3 independent immunoblot assays. B HPLC-MS/MS quantification of the total ceramides, the specified ceramide species. Shown (mean ± SD) is one representative of three independent experiments that were quantified in triplicates. C With a final molar ratio of 6.5 PA into 1 FF-BSA, transfected cells were replaced with fresh medium containing 0, 50 or 250 μM of PA 48 h later for indicated periods. 0 μM represents the medium containing 0.25% of FF-BSA and NaOH. Shown is one representative of three independent experiments for determining the transcriptional levels of CERS2, CERS6 and DEGS1 in differently treated cells. Immunoblot analysis determined CERS2 and CERS6 when the cells were treated with 250 μM of PA for 24 h. D The cells (in triplicates) were treated with medium containing 250 μM PA alone, or inclusion of 5 μM Myr for 24 h and analysed using HPLC-MS/MS. The control medium contained 0.25% of FF-BSA, NaOH and DMSO. Shown is one representative of three independent experiments. Data (mean ± SD) represent the increased folds of total ceramides and the indicated ceramide species relative to that of empty-vector-transfected HepG2 cells cultured with control medium. The differences were compared with one-way ANOVA analysis. $P < 0.05, $$P < 0.01, $$$P < 0.001 between Ref-HBV and empty-vector; *P < 0.05, **P < 0.01, ***P < 0.001 between Ref-HBV and mtPreS1 or mtPreS2; #P < 0.05, ##P < 0.01, ###P < 0.001 between mtPreS1 and mtPreS2

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