Figure 12. Abrogation of the UPR-autophagy–mediated suppression of IFN-β activation by knockdown of LAMP2 and Rab7.
(A) Huh7/mRFP-GFP-LC3 cells were transfected with 400 pmol each of control, LAMP2, or Rab7 siRNA duplexes for 48 hours, and cells were quantified for RFP-LC3– (RFP-positive) and RFP-GFP-LC3–labeled (RFP-GFP-positive) puncta structures by confocal microscopy (bottom panel). The yellow-colored image represents the RFP-GFP-LC3–labeled puncta structure. A set of representative confocal images is also shown (top panel; scale bars: 10 μm). (B) Huh7/mRFP-GFP-LC3 cells were transfected with 400 pmol each of the indicated siRNA duplexes. Forty-eight hours later, cells were transfected with the pIFN-β/Fluc promoter reporter and cultured for 24 hours. The cells were then transfected with control HCV 5′-UTR RNA or 3′-UTR PAMP RNA and maintained for an additional 12 hours. The cells were left untreated or treated with EBSS or tunicamycin for 6 hours. Cells were then analyzed for IFNB promoter reporter activation (left panel). A set of cells 48 hours after siRNA transfection was analyzed by the Western blotting (right panel). (C and D) Huh7/mRFP-GFP-LC3 cells were infected with HCV at an MOI of 10 and cultured for 6 days. The cells were then transfected with the indicated siRNA duplexes as described in A. Seventy-two hours after transfection, cells were harvested and analyzed for the intracellular HCV RNA level (C) and analyzed for the indicated proteins by Western blotting (D). Data represent mean ± SEM (n = 3) (A–C).