Journal: eLife

Article Title: Differences across cyclophilin A orthologs contribute to the host range restriction of hepatitis C virus

doi: 10.7554/eLife.44436

Figure Lengend Snippet: ( A ) Schematic of the engineered murine hepatoma cell line, Clone 8 + murine ApoE (mApoE) derived from Hep56.1D cells. ( B ) SEC14L2 expression verified by western blot. Mouse anti-human SEC14L2 shown in green (~45 kDa) and rabbit anti-β actin shown in red (~42 kDa). ( C ) miR-122 expression was assessed by RT-qPCR. Results are normalized to U6 and shown as fold change relative to Huh7.5 cells (set at 100). ( D ) Clone 8 ± ApoE express human CD81, Venus YFP-human OCLN, Cerulean-human CLDN1, mKate2-human SRBI and murine ApoE-tagRFP as assessed by flow cytometry. ( E ) The kinetics of HCV replication were assessed in Clone 8 ± ApoE, the parental Hep56.1D cells and the highly permissive human hepatoma cell line Huh7. Cells were infected with Jc1-Gluc at an MOI of 0.1 and supernatants collected daily for six days. On even-numbered days as indicated by the labeled arrows, cells were washed with PBS and the media changed to assess de novo replication. Lines and error bars represent the mean ± SD.

Article Snippet: Huh7 cells were obtained from the American Tissue Culture Collection (ATCC) and Hep56.1D cells from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Derivative Assay, Expressing, Western Blot, Quantitative RT-PCR, Flow Cytometry, Infection, Labeling

Journal: eLife

Article Title: Differences across cyclophilin A orthologs contribute to the host range restriction of hepatitis C virus

doi: 10.7554/eLife.44436

Figure Lengend Snippet: ( A ) Clone 8 + ApoE cells were transduced with mouse, human, triply murinized human, or triply humanized mouse CypA in bicistronic lentiviral constructs expressing eGFP. Transduction efficiency assessed by flow cytometry. ( B ) The transduced Clone 8 cells were infected with Jc1-Gluc (MOI = 0.1) and the media changed daily. The different factors transduced into the Hep56.1D cells are specified below the graph. Note that the "—*” for mApoE indicates that these cells were not transduced to express exogenous mApoE; their endogenous expression was not disrupted. Results shown are from two representative experiments, each with triplicate samples. Gaussia luciferase activity was assessed in these supernatants as a proxy for HCV replication and is expressed as relative luminescence units (RLU). Ordinary one-way ANOVA test performed on the Hep56.1D and derivative lines followed by Tukey’s multiple comparisons test with a single pooled variance. The dashed line labeled ‘background’ is the average signal from supernatants of naïve cells. ( C ) As in ( B ), Huh7 cells, Hep56.1D and derivative lines as shown on the x-axis were infected with Jc1-Gluc (MOI = 0.1) and supernatants collected at six dpi to infect naïve Huh7.5 cells. Following infection of these Huh7.5 cells with the supernatants, the media was changed daily. Shown is the Gaussia luciferase activity of the Huh7.5 supernatants three dpi with the origin of the supernatants used for the original infection indicated on the X axis. Lines and error bars represent the mean ± SD. The dashed line labeled ‘background’ is the average signal from supernatants of naïve cells. Ordinary two-way ANOVA test performed on the Hep56.1D and derivative lines followed by Dunnett’s multiple comparison test, with all means compared to that of Hep56.1D. Lines and error bars represent the mean ± SD. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Article Snippet: Huh7 cells were obtained from the American Tissue Culture Collection (ATCC) and Hep56.1D cells from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Transduction, Construct, Expressing, Flow Cytometry, Infection, Luciferase, Activity Assay, Labeling, Comparison

Journal: eLife

Article Title: Differences across cyclophilin A orthologs contribute to the host range restriction of hepatitis C virus

doi: 10.7554/eLife.44436

Figure Lengend Snippet:

Article Snippet: Huh7 cells were obtained from the American Tissue Culture Collection (ATCC) and Hep56.1D cells from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Flow Cytometry, Expressing, shRNA, Cloning, Mutagenesis, Luciferase, Recombinant, Plasmid Preparation, Sequencing, Software