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. 2015 Jul 17;10(7):e0132839. doi: 10.1371/journal.pone.0132839

Fig 4. AAM-B is required for the virion production without affecting lipid droplet formation.

Fig 4

(A) Huh7 cells were infected with Jc1 for 4 h. At 48 h postinfection, cells were further transfected with the indicated siRNAs. At 24 h after siRNA transfection, the culture medium was replaced with fresh medium and cells were further grown for the indicated time points. AAM-B mRNA levels (A), intracellular HCV RNA levels (B), and extracellular HCV RNA levels (C) were quantified by qPCR. (D) (Left) Huh7 cells were infected with Jc1 for 4 h. At 48 h postinfection, cells were further transfected with the indicated siRNAs. At 24 h after siRNA transfection, the cells were transfected with plasmid expressing either wild-type AAM-B or siRNA resistant mutant AAM-B. At 48 h after plasmid transfection, extracellular HCV RNAs were quantified by qPCR. pAAM-B WT, V5-tagged wild-type AAM-B; pAAM-B SR, V5-tagged siRNA resistant mutant AAM-B. (Right) Huh7 cells were treated as described in the left panel and protein expressions were analyzed by immunoblot analysis using the indicated antibodies. (E) Naïve Huh7 cells were infected with culture supernatant harvested from (A) for 4 h. At 48 h postinfection, intracellular HCV RNA levels were analyzed by qPCR. (F) Huh7 cells were infected with Jc1 for 4 h. At 48 h postinfection, the cells were transfected with either negative or AAM-B specific siRNAs. At the indicated time points, the cells were harvested and washed twice with PBS. The cells were subjected to three consecutive freeze-thaw cycles and then centrifuged for 30 min in a microfuge. Supernatant was used as intracellular Jc1. Naïve Huh7 cells were infected with supernatant for 4 h. At 48 h postinfection, relative intracellular HCV infectivity was determined by qPCR. (G) Huh7 cells were infected with Jc1 for 4 h. At 48 h postinfection, cells were further transfected with the indicated siRNAs. The cells were fixed and incubated with BODIPY for 1 h at 37°C. Cells were counterstained with 4',6-diamidino-2-phenylindole(DAPI) to label nuclei. Immunofluorescence images were processed using the Zeiss LSM 700 laser confocal microscopy system. Bars, 10μm.