PMC:7464876 / 12331-14444 JSONTXT

{"project":"2_test","denotations":[{"id":"32806600-24614091-20296197","span":{"begin":1080,"end":1082},"obj":"24614091"},{"id":"32806600-32017353-20296198","span":{"begin":1083,"end":1085},"obj":"32017353"},{"id":"32806600-20406418-20296199","span":{"begin":1365,"end":1367},"obj":"20406418"},{"id":"32806600-24237698-20296200","span":{"begin":1368,"end":1370},"obj":"24237698"}],"text":"2.2. SiRNAs, Cell Culture and Transfection\nFor transient expression analyses, the human hepatocellular carcinoma cell line HuH-7 was used that was obtained by the European Collection of Authenticated Cell Cultures (http://cellbank.nibiohn.go.jp/english/). This cell line is not susceptible to HBV infection, because it expresses very low levels of the NTCP receptor and is therefore a useful model to study the production and release of the virus rather than infection [7]. Cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 5-µg/mL ciprofloxacin (Fresenius Kabi, Bad Homburg, Germany). Transfections of cells with plasmid DNAs were performed with Lipofectamine™ Plus (Thermo Fisher Scientific, Waltham, MA, USA). For depletion of EAP20 (NM_032353.3), Sar1A (NM_01142648), Sar1B (NM_016103.3), Sec23A (NM_006364.4), Sec23B (NM_006363), Sec24A (NM_021982) or Sec24B (NM_006323), single siRNA duplexes or siGENOME SMARTpool RNAs (Dharmacon, Lafayette, CO, USA) were used as described [22,29]. To silence the expression of ERGIC-53 (NM_005570), a siRNA (5′-GGACAGAAUCGUAUUCAUC-3′) targeting nucleotide positions 1009–1027 was obtained from Dharmacon (Dharmacon, Lafayette, CO, USA). The specificity and efficacy of this siRNA has been approved in independent studies [37,38,39]. A control siRNA with no known homology to mammalian genes was purchased from Qiagen (Qiagen, Hilden, Germany). For combined transfection, HuH-7 cells were first transfected with siRNAs by using the Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). In a typical experiment, 3 × 105 cells per well of a 12-well plate were transfected with a final concentration of 20-nM siRNA, according to the protocol of the supplier. After 48 to 72 h, cells were retransfected with 2-µg plasmid DNA, and transfected cells were harvested after an additional 48 to 72 h, as indicated in the text. For drug treatment, cells were incubated with 1-µM brefeldin A (BFA; Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C."}