*, P0.05; **, P0.005; ***, P0.0005; NS, not significant. (TIF) Click here for additional data file.(3.0M, tif) S5 FigTransient overexpression of MAVS variants induces comparable levels of type I and III IFN as well as ISGs in A549 cells. as loading control. Empty control refers to cells transduced with the empty vector. (D) Huh7 cells expressing the inactive mutant (NS3/4A-S139A; left panel) or the parental protease (right panel) were transfected with expression constructs specified on the left and 24 hours later cells were fixed and analyzed by fluorescence microscopy. Nuclei were stained with DAPI. Scale bar, 20 m.(TIF) ppat.1005264.s002.tif (4.6M) GUID:?83EE98AF-404C-4E8F-9CA7-6A5C44669D02 S3 Fig: No difference in the activation of the IFN response by peroxisomal and mitochondrial MAVS in a type I IFN competent cell line. (A) A549-NS3/4A expressing cells were transduced with lentiviruses containing non-cleavable wt-, pex- or mitoMAVSCR as well as Pex14-HA that served as control. Cells were lysed 36 hours after transduction and expression of MAVSCR, endogenous cleaved MAVS and MxA were determined by Western blot using antibodies specified in the right of each panel. The relative expression levels of MAVS normalized Evobrutinib to GAPDH and wtMAVSCR (set to 1) are shown in the right panel (mean values of three independent experiments and standard errors). (B-C) Cells were stimulated by overexpressing MAVS variants. (B) To determine activation of the IFIT1, IFNB and promoter (left, middle and right panel, respectively), cells were transfected with firefly luciferase reporter plasmids and a SV40-based Renilla luciferase plasmid to normalize for transfection Evobrutinib efficiency. After 24 hours cells were lysed and luciferase activity was measured. (C) qRT-PCR analysis was performed to determine mRNA levels for the genes specified in the top of each panel. Values were normalized to GAPDH by using the ct method. Data represent the mean from four independent experiments. Bars indicate the standard error. *, P0.05; **, P0.005; ***, P0.0005; NS, not significant.(TIF) ppat.1005264.s003.tif (3.0M) Evobrutinib GUID:?A4D1A3F4-A8C1-4C9E-B557-717291DF3172 S4 Fig: Transient overexpression of MAVS variants in 293T cells and activation of type I and III IFN response. (A) 293T cells stably expressing NS3/4A were transduced with lentiviral vectors encoding for non-cleavable wt-, pex- or mitoMAVSCR as well as Pex14-HA that served as negative control. Expression of transduced IMPA2 antibody genes was determined by Western blot. The quantification in the right panel displays MAVS expression levels normalized to -actin and wtMAVSCR that was set to one. Mean values and standard error of three independent experiments are given. (B) Thirty six hours after transduction cell culture supernatants were collected to measure IFN-1C3 protein levels by ELISA. Dashed line represents the limit of detection (LOD). N.d., not detectable. (C) To measure promoter activation firefly luciferase reporter plasmids specified on the top of each panel were co-transfected with a SV40-based Renilla luciferase plasmid. Values were normalized to those obtained for Pex14-HA (set to 1). (D) Amounts of mRNAs specified in the top of each panel were quantified by qRT-PCR. All data were normalized to the housekeeping gene GAPDH using the ct method. Bars indicate the standard error. All experiments were performed at least three times independently. *, P0.05; **, P0.005; ***, P0.0005; NS, not significant.(TIF) ppat.1005264.s004.tif (3.0M) GUID:?E3185C31-98C2-49A8-8EB1-952A4F460F1D S5 Fig: Transient overexpression of MAVS variants induces comparable levels of type I and III IFN as well as ISGs in A549 cells. (A) A549 and (B) A549-MAVSKO cells generated by a CRISPR/Cas9 approach were transduced with lentiviral.