FIG 6.

Seed sequence-matched siRNA shows that MOBKL1B is not required for HCV RNA replication in Huh-7.5 cells. (A) Three MOBKL1B-specific siRNAs (1, 2, and 3), their seed sequence-matched control siRNAs (1 C911, 2 C1012, and 3 C911), or irrelevant (IRR) siRNA was transfected into Huh-7.5 cells as described for Fig. 2. Depletion of endogenous MOBKL1B protein was analyzed by Western blotting. β-Actin levels are shown as a control. (B) MOBKL1B siRNA treatment is not cytotoxic. Huh-7.5 cells were transfected twice with three individual MOBKL1B-specific siRNAs (1, 2, and 3), their seed sequence-matched siRNAs (1 C911, 2 C1012, and 3 C911), or irrelevant (IRR) siRNA at 24-h intervals. Toxicity of the MOBKL1B knockdown was evaluated with a luminescent cell viability assay (mean of n = 3; error bars, SD). RLU, relative light units. (C) MOBKL1B depletion does not inhibit HCV genome replication. Depletion of MOBKL1B in CD81 knockdown Huh-7.5 cells was performed, as described above. Jc1FLAG(p7-nsGluc2A) was transfected at 48 h after the second siRNA transfection. Medium was collected 48 h later for luciferase quantitation. (D) MOBKL1B depletion and infectious virus production. Naive Huh-7.5 cells were infected from the media from cells treated with IRR, siRNA 3, and siRNA 3 C911 (shown in panel C), and luciferase was measured to analyze infectious virus production. Values are normalized to RLU measured in irrelevant siRNA-treated cells (mean of n = 3; error bars, SD).