Incubations were performed at concentrations indicated in the figures and figure legends. Experiments were performed at least in triplicate. CoPP was purchased from Frontier Scientific Europe
www.selleckchem.com/products/pd-0332991-palbociclib-isethionate.html Ltd., Carnforth, Lancashire, UK). Methylene chloride (MC), lactoferrin, deferoxamine, and FeCl3 were purchased from Sigma Aldrich GmbH (Steinheim, Germany). Biliverdin was purchased from MP Biomedicals (Heidelberg, Germany). To verify altered gene expression, RNA was transcribed into complementary DNA by using the Verso cDNA Kit (Thermo Fisher Scientific, Waltham, MA). Oligonucleotides for subsequent polymerase chain reaction (PCR) reactions were obtained from Metabion International AG (Martinsried, Germany). Oligonucleotide pairs for real-time reverse transcription (RT)-PCR are summarized in Table 1. Real-time RT-PCR was performed by using
the CFX Real-Time system (BIO-RAD, Munich, Germany) and reagents from Abgene (Thermo Fisher Scientific, Germany). Reactions were performed in a 10-μL volume. To confirm amplification specificity, PCR products were subjected to melting curve analysis and gel electrophoresis. Fifteen micrograms protein were fractionated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Western blots were developed using an Trametinib in vitro enhanced chemiluminescence system (Amersham, Freiburg, Germany) according to the manufacturer’s instructions. Semiquantitative evaluation was performed using the VersaDoc Imaging System (BioRad Laboratories GmbH, Munich, Germany). Antibodies for western blots were rabbit anti-HO-1 (Stressgen Biomol, Hamburg, Germany), mouse anti-hepatitis
C NS5 (MorphoSys UK Ltd., Oxford, UK), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase MCE (HyTest Ltd., Turku, Finland). Luciferase activity of LucUbiNeo-ET replicon cells was measured using the Luciferase Assay System (Promega, Mannheim, Germany) and normalized to the protein content in the individual samples. For all luciferase assays shown, protein contents in lysates were comparable, indicating that incubations did not affect cell metabolism. The results were analyzed using Student t test if two groups were compared and the Dunnett’s test if more groups were tested against a control group. If variances were not homogeneous in the Student t test, the results were analyzed using the Welsh test. All data in this study are expressed as a mean ± standard error of the mean. P ≤ 0.05 was considered significant. HO-1 overexpression has recently been shown to interfere with HCV replication.25, 26 To define the impact of HO-1 on HCV replication more precisely, Huh-5-15 replicon cells and their parental cell line Huh-7 (Fig. 1), as well as LucUbiNeo-ET replicon cells (Fig. 2), were incubated in the presence of the HO-1 inducer CoPP. Measurement of HCV polyprotein expressions by real-time RT-PCR showed that HCV replication was dose-dependently impaired (Fig.