Journal: Hepatology (Baltimore, Md.)

Article Title: Role of low-density lipoprotein receptor in the hepatitis C virus life cycle.

doi: 10.1002/hep.25501

Figure Lengend Snippet: Fig. 2. Kinetics of virus internalization in comparison to lipopro- teins. (A) HCVcc and HCVpp infections were synchronized by binding for 1 hour at 4C. Internalization was then allowed by shifting the tem- perature to 37C. At different time points, cell-bound particles were removed by trypsin treatment. Infectivity was determined after 48 hours by measuring the luciferase activity. Results are presented as the means of three independent experiments 6 SD. The 100% value is set as the maximal luciferase activity value after 240 minutes of internalization. HCVcc and HCVpp internalizations are shown as solid and dashed lines, respectively. (B) DiI-LDL and DiI-IDL (10 lg/mL) were bound to cells for 1 hour at 4C. Internalization was then allowed by shifting the temperature to 37C. At different time points, the reac- tion was stopped and noninternalized lipoproteins were removed from the cell surface by heparin treatment and cells were analyzed by flow cytometry. The data from three independent experiments are shown as the mean of percentage of the maximum value 6 SD. DiI-LDL and DiI-IDL internalizations are shown as solid and dashed lines, respec- tively. The fluorescence decrease observed at later time points was likely caused by lipoprotein degradation. SD, standard deviation.

Article Snippet: Human IDL and LDL were from Athens Research & Technology, Inc. (Athens, GA).

Techniques: Virus, Comparison, Binding Assay, Infection, Luciferase, Activity Assay, Cytometry, Standard Deviation

Journal: Hepatology (Baltimore, Md.)

Article Title: Role of low-density lipoprotein receptor in the hepatitis C virus life cycle.

doi: 10.1002/hep.25501

Figure Lengend Snippet: Fig. 3. LDLR plays a role in HCV replication. (A) Huh-7 cells were incubated for 2 hours with DiI-LDL or DiI-IDL (10 lg/mL) at 37C in the presence (gray lines, C7) or absence (black lines, control) of mAb C7 (10 lg/ml). Cells were then processed for flow cytometry analysis. Cells processed in the absence of lipoproteins are shown as filled gray fields (Neg). Mab C7 reduced LDL and IDL binding to by 78% and 87%, respectively. (B) Huh-7 cells were infected with HCVcc in the presence or absence of Mab C7 (5 lg/mL) and were further incubated in the pres- ence or absence of mAb C7, as indicated in the table. Infectivity was determined after 48 hours by measuring luciferase activity. ON, overnight incubation. *P < 0.05; **P < 0.01. (C) Huh-7 cells were electroporated with viral RNA (HCV or SINV) and then incubated with mAb C7 or a nonspecific antibody at a concentration of 5 lg/mL. At different times postelectroporation, replication was determined by measuring luciferase activity. A nonreplicating HCV GND mutant was used as a negative control of replication. Results are shown as the mean for three independent experiments 6 SD. Of note, the drop in luciferase expression did not seem to be the result of an effect on translation, because the luciferase values did not differ at 4 hours after electroporation. (D) Analysis of Huh-7 lipid composition in the presence of mAb C7. Neutral and phospho- lipid cell content was tested, as described in Materials and Methods. The total amount of neutral lipid/phospholipid was set to 100%. Content of different lipids was calculated as the percentage of the total lipid amount. TG, triglycerides; SM, sphingomyelin; PS, phosphatidylserine; PI, phosphatidylinositol; SD, standard deviation.

Article Snippet: Human IDL and LDL were from Athens Research & Technology, Inc. (Athens, GA).

Techniques: Incubation, Control, Cytometry, Binding Assay, Infection, Luciferase, Activity Assay, Concentration Assay, Mutagenesis, Negative Control, Expressing, Electroporation, Standard Deviation