Lumistar optima luminescence counter

For Gaussia luciferase, the secreted luciferase activity in the cell culture medium was measured using a BioLux^®Gaussia Luciferase Flex Assay Kit (New England Biolabs). Gaussia luciferase activity was quantified using a LumiStar Optima luminescence counter (BMG LabTech, Offenburg, Germany). For the full-length infectious models (HEV-p6), intracellular viral RNA was quantified. RNA was isolated using a Machery-Nucleo Spin RNA II kit (Bioke, Leiden, The Netherlands) and quantified using a NanoDrop ND-1000 spectrophotometer (Wilmington, DE, USA). cDNA was prepared from total RNA using a cDNA Synthesis Kit (Takara Bio Inc, USA). The HEV RNA level was quantified using a SYBR Green–based real-time PCR assay (Applied Biosystems® SYBR® Green PCR Master Mix, Life Technologies, CA, USA) according to the manufacturer’s instructions. The PCR steps consisted of a 10 min holding stage (95 °C) followed by 40 cycles of 15 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene to normalize gene expression. Relative gene expression was normalized to GAPDH using the formula 2^−ΔΔCT (ΔΔCT = ΔCT_sample − ΔCT_control). The HEV primer sequences were as follows: HEV-F, 5’-ATTGGCCAGAAGTTGGTTTTCAC-3’; HEV-R, 5’-CCGTGGCTATAATTGTGGTCT-3’; GAPDH-F, 5’-TGTCCCCACCCCCAATGTATC-3’; GAPDH-R, 5’CTCCGATGCCTGCTTCACTACCTT-3’.

An ATP Bioluminescence Assay Kit HS II was used to measure the ATP content of cells according to the manufacturer’s instructions (Roche Life Science, Penzberg, Germany). Cells were seeded into a 96-well plate with or without mETC complex inhibitors and cultured for 48 h. Then, cells were harvested and suspended in dilution buffer at a concentration of 1 × 10⁵ /ml. The same volume of cell lysis reagent was added into to above cell suspension and incubated at 15 °C for 5 min and for an extra 2 min at 100 °C. Subsequently, the cell suspension was centrifuged at 10,000 g for 60 s and the supernatant was transferred to a fresh tube. Samples were kept on ice until measurement. A mixture of 50 ul luciferase reagent was added to 50 ul supernatant or standards provided by ATP Assay kit. Luminescence was detected after 1 s delay using a microplate reader (LumiStar Optima Luminescence Counter, BMG Labtech, Offenburg, Germany) (excitation = 535 nm; emission = 587 nm).