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. 2015 Apr 8;89(12):6294–6311. doi: 10.1128/JVI.03631-14

FIG 2.

FIG 2

The impact of Xrn2 knockdown on miR-122-dependent and miR-122-independent subgenomic HCV replication. (A) Hep3B cells were electroporated with siXrn2 or siControl for preknockdown and 3 days later (day 0) were electroporated again with the indicated siRNAs, miRNAs, mRNAs, and wild-type HCV SGR RNAs as described for Fig. 1B. “Dependent” samples were electroporated with miR-122, while “independent” samples were electroporated with miControl. (B) The effect of Xrn2 knockdown on miR-122-dependent and -independent replication was evaluated as described in the legend to Fig. 1C. Significance was determined by unpaired parametric t test. (C) The effectiveness of siXrn2 at reducing Xrn2 protein levels in Hep3B cells was determined by Western blotting with antibodies against Xrn2 and β-actin. A representative blot is shown depicting Xrn2 protein levels 3 days post-first electroporation (at the time of second electroporation, which includes viral RNA and miRNAs) in the first two lanes and 6 days post-first electroporation or 3 days post-second electroporation. Percent knockdown ± standard deviation relative to the siControl-treated cells was determined by infrared band quantification on blots from three independent experiments. (D) Huh7.5 cells were treated with siXrn2 as described in the legend to Fig. 1F. “Dependent” samples were electroporated with wild-type SGR RNA (depicted above), while “independent” samples were electroporated with mutant SGR S1+S2:p3 viral RNA, which does not respond to miR-122, as depicted in Fig. 1E. (E) The effect of Xrn2 knockdown on miR-122-dependent and -independent replication was evaluated as described in the legend to Fig. 1G. Significance was determined by paired parametric t test. (F) The effectiveness of siXrn2 in reducing Xrn2 protein levels in Huh7.5 cells was determined as described in the legend to panel C, and a blot representative of three independent experiments is shown. (G) The effect of siXrn2, viral RNAs, and microRNAs on cell survival was evaluated by WST-1 3 days postelectroporation from samples in panels A and D, and cell numbers are normalized to siControl-miControl or siControl-SGR S1+S2:p3 samples. (H) Transfection efficiency in the experiments in panels A and D was evaluated 2 h postelectroporation by measuring Renilla luciferase expression from a coelectroporated mRNA. Samples are normalized to siControl-miControl or siControl-SGR S1+S2:p3 samples. (I) Untreated Hep3B and Huh7.5 cell lysates were analyzed by using Western blotting to show steady-state levels of Xrn2 protein in each cell type.