PLIC-1 Inhibits TLR3 Activation Figure 5. PLIC-1 co-localized with Trif. The human hepatoma cells Huh7.5.1, were seeded on glass cover slip and then transfected with 0.1 mg of YFP-PLIC-1 alone (A) or with 0.1 mg Flag-Trif (B) in a 24-well plate format. 24 hours post-infection, cells were fixed, permeabilized and stained with anti-Flag M2 antibody. Confocal and differential interference contrast (DIC) images were taken with a Zeiss Meta LSM510 microscope. C. 0.1 mg of GFP expression plasmid was transfected with 0.1 mg Flag-Trif for confocal imaging. D. 0.1 mg of YFP-PLIC-1 expression plasmid was transfected with 0.1 mg Flag-MAVS into Huh7.5.1 cells for confocal imaging. doi:10.1371/journal.pone.0021153.g005 contents [45,46]. In this case, portions of cytoplasm and cell organelles are first taken into phagosomes that fuse with lysosomes into phagolysosomes in which proteins will be finally degraded by lysosomal enzymes [47]. Since both PLIC-1 and Trif can localize to autophagosomes and the PLIC-1-dependent effect on Trif is sensitive to Nocodazole treatment, it is tempting to envision that cells may utilize the authophagic pathway that typically degrades cellular components during starvation to degrade Trif signalosome and hence terminate the Trif-dependent innate signaling. Future work is warranted to explore this novel mechanism in detail. Materials and Methods Cell Lines and reagents 293T (ATCC CRL-11268) cell line and the human hepatoma cell line Huh7.5.1 (gift from Dr. F. Chisari) were cultured in DMEM (Invitrogen Life Technologies) supplemented with 10% FBS (HyClone), 1% penicillin, and 10 mg/ml streptomycin. The mouse macrophage cell line J774 was a generous gift from Dr. David Hackam (University of Pittsburgh School of medicine, PA). J774 cell line was maintained in DMEM supplemented with 5% penicillin and streptomycin, 1% NEAA, and 10% fetal bovine serum (Hyclone). The human lung cancer cell line A549 (ATCC CCL-185) was maintained in F12K media (ATCC 30-2004) supplemented with 10% fetal bovine serum and 5% penicillin and streptomycin. TLR4/MD2 stable cell line was obtained from Dr. D. Golenbock (University of Massachusetts, Worcester). HEK/ TLR7 cell line was a generous gift of Dr. S. Saumendra (University of Pittsburgh). Anti-HA, anti-Flag M2, and anti- PLIC-1 (U7383) antibodies were purchased from Sigma. DNA constructs PLIC-1 N terminal and PLIC-2 full length plasmids were obtained from the Howley lab (Boston). The NF-kB-Luc plasmid Figure 6. PLIC-1 and Trif co-localized with LC3. The human hepatoma cell line Huh7.5.1 cells were seeded on glass cover slip and then transfected with 0.1 mg of YFP-PLIC-1 and RFP-LC3 (A), or 0.1 mg YFP-Trif and RFP-LC3 in HEK (B) or Huh7.5.1 (C) cells in a 24-well plate format. 24 hours post-infection, cells were fixed and imaged using a Zeiss Meta LSM510 microscope. In all figures, co-localization was indicated as yellow dots in the merged images. doi:10.1371/journal.pone.0021153.g006 PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e21153