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. Author manuscript; available in PMC: 2012 Sep 4.
Published in final edited form as: Cell Host Microbe. 2011 Jan 20;9(1):32–45. doi: 10.1016/j.chom.2010.12.002

Fig. 3. Effect of PI4KIIIα and PI4KIIIβ silencing on HCV replication and colocalization of these lipid kinases with NS5A.

Fig. 3

(A, B) Cells containing a genotype (gt) 2a or 1b luciferase reporter replicon specified in the top of each panel were transfected with siRNAs targeting PI4KIIIα or PI4KIIIβ (three individual siRNAs each) or with the positive control siRNA (HCV321) or the negative control siRNAs (DV-3’NTR or NegC#1). SiRNAs were transfected twice and RNA replication was determined 48 h after the second transfection by luciferase assay. RLUs were normalized to the mean of the negative controls. Data (mean +/− SD; n = 3 in triplicates) were analyzed using a one-way t-test. (C) Lysates prepared from Con1ET replicon cells 48 h after the second transfection were analyzed by immunobloting for PI4KIIIα, PI4KIIIβ, Transferrin receptor (TfR) and NS5A. Levels of NS5A were quantified and normalized to the corresponding TfR levels. Values are given in % relative to the mean of the NS5A levels of the negative control treated cells. (D, E) Huh7-Lunet cells (mock) or cells containing a selectable JFH-1 replicon (neo-sgJFH-1) were stained with a NS5A- (red) and either a PI4KIIIα- (D) or a PI4KIIIβ-specific antibody (green) (E). Nuclear DNA was stained with DAPI (blue). An enlargement of the sections indicated by a white square in each of the merged images is shown in the corresponding crop panel in the bottom. Images were acquired with a confocal microscope.