MG132 was included in the assays to prevent vFLIP degradation. in WT and KO BCBL-1 (1A4) cells untreated and treated with 10 M zVAD-fmk for 1 day. The cells were seeded at 2×105 cells/ml. (B) HHV-8 effective replication assay. HHV-8 viral genomes were purified from your tradition supernatants of WT (C6) and KO (1A4 and 3B11) BCBL-1 cells cultivated under high-density tradition for 2 days and subjected to quantitative PCR to determine the copy quantity of the viral genome. Data are displayed as mean SD of triplicate samples. (C) The cells were incubated in EBSS for 6 h or treated with rapamycin (Rapa), 50 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), 100 nM staurosporine (STS), 10 M carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and 5 M rotenone (Rot) in total media for 1 day. Cell viability was assessed by using CellTiter-Glo?. Data are displayed as mean SD of two self-employed experiments in triplicate. (*p 0.005 and **p 0.05).(TIF) ppat.1007058.s002.tif (953K) GUID:?96772A4C-9490-4BA2-8AD7-AC25A0A16D58 S3 Fig: p62/SQSTM1 expression in WT and KO BJAB and AKATA cells. Immunoblotting was performed with components derived from the BJAB and AKATA Rabbit polyclonal to COT.This gene was identified by its oncogenic transforming activity in cells.The encoded protein is a member of the serine/threonine protein kinase family.This kinase can activate both the MAP kinase and JNK kinase pathways. cells cultured at different densities, low (5×104 cells/ml) and high (2×105 cells/ml), for 2 days.(TIF) ppat.1007058.s003.tif (315K) GUID:?AA452773-C275-411A-9355-E2FDEC0EDC8C S4 Fig: Effect of epitope tagging about basal and MAVS-induced vFLIP stability. Components from 293T cells transfected with (±)-WS75624B the indicated epitope tagged and non-tagged vFLIPs together with or without Flag-MAVS, for 24 h were separated by SDS-PAGE and immunoblotted with anti-vFLIP, Flag, and -actin antibodies.(TIF) ppat.1007058.s004.tif (362K) GUID:?079DCB2B-64CD-4D76-A25F-972FD90E1124 S5 Fig: Real time-qPCR analysis of V5-vFLIP expression in TRAF6-cotransfected cells. Total RNAs were isolated from WT and KO 293T cells co-transfected with pICE_V5-vFLIP plasmid together with the indicated amounts of Flag-TRAF6 plasmid for 24 h and subjected to actual time-qPCR. The relative mRNA manifestation of V5-vFLIP normalized to 18S RNA was determined by comparison to control (WT cells transfected with V5-vFLIP without TRAF6) and depicted in the column graph. Data are displayed as mean SD of triplicate samples. NS indicates not significant (p 0.1).(TIF) ppat.1007058.s005.tif (322K) GUID:?40F92DFD-285A-4717-9732-AE9836F77128 S6 Fig: TRAF6 partially localizes to peroxisomes inside a MAVS-dependent manner. Triple immunostaining with antibodies to Flag (TRAF6), MAVS, and PMP70 in WT and KO 293T cells transfected with Flag-TRAF6 together with or without MAVS-Pex. Fluorescent images were merged with an image of DAPI. The inset boxes in the merged images were zoomed in to the right side of the images. Yellow dots show localization of TRAF6 to peroxisomes and white dots show co-localization of TRAF6 and MAVS on peroxisomes. Scale bar shows 10 m.(TIF) ppat.1007058.s006.tif (3.6M) GUID:?0A0C5295-CEA2-4516-BD96-6739956154E2 S7 Fig: Peroxisomes are required for MAVS-induced vFLIP stabilization. Triple immunostaining with antibodies to Flag (MAVS), V5, and PMP70 in WT and KO 293A cells transfected with V5-vFLIP WT or mPTSX together with Flag-MAVS, Flag-MAVS-Mito, and Flag-MAVS-Pex. Fluorescent images were merged with an image of DAPI. The inset boxes in the merged images were zoomed in at the bottom of the number. Yellow dots show localization of vFLIP to peroxisomes and white dots show co-localization of vFLIP and MAVS on peroxisomes. V5-vFLIP was barely recognized in KO cells, and V5-vFLIP mPTSX was barely recognized in WT and KO cells. Scale bar shows 20 m.(TIF) ppat.1007058.s007.tif (4.9M) GUID:?4529FAD8-CC93-442E-80E3-D8A99A234BD6 S8 (±)-WS75624B Fig: The effect of cell-penetrating versions of vFLIP-derived peptides on MAVS-induced vFLIP stabilization. (A) Sequences of TAT and TAT-fused vFLIP peptides. (B) Immunoblotting with components of 293A cells co-transfected with V5-vFLIP and bare (CMAVS) or Flag-MAVS (+ MAVS) vectors and then treated with the peptides for 1 day.(TIF) ppat.1007058.s008.tif (457K) GUID:?B71EC397-A4CC-4BE9-B30C-C8FA7A251796 S9 Fig: The effect of the vFLIP peptide 2H1 on MAVS-induced antiviral responses. (A-B) Reporter assays in (±)-WS75624B 293T cells transfected with bare (CMAVS) or Flag-MAVS (+ MAVS) vectors along with IFN–Luc (A) or NF-B-Luc (B) (±)-WS75624B reporter in the presence of TAT and TAT-2H1 peptides for 1 day. Data are offered as mean SD of triplicate samples. NS indicates not significant. (C) Immunoblots of components of 293T cells transfected with bare vector or MAVS plasmid in the presence of TAT and TAT-2H1 peptides for 1 day.(TIF) ppat.1007058.s009.tif (517K) GUID:?F20FCD8C-B8F1-4619-9FD8-42499AD7B0F2 S10 Fig: K118-only vFLIP is practical. Reporter assay was performed in 293T cells transfected with bare vector, vFLIP, or K118-only vFLIP vectors along with NF-B-Luc (B) reporter plasmid for 1 day. Data are offered as mean SD of triplicate samples.(TIF) ppat.1007058.s010.tif (185K) GUID:?2C8BC882-4D61-4702-9698-7036BDE59B90 S11 Fig: vFLIP degradation from the 2H1 peptide does not affect HHV-8 reactivation..