(A) Schematic representation of YOD1 overexpression constructs. YOD1 WT or C160S and GFP were co-expressed using T2A site under the control of EF1α promoter, which in turn is DOX/tTR-KRAB-controlled. (B) YOD1 WT and YOD1 C160S are overexpressed upon doxycycline (DOX) treatment of lentivirally transduced HeLa cells. Transduced cells were grown in DOX containing medium for 72 hr and after cell lysis subjected to Western Blotting. (C) YOD1 WT (left panel) or C160S (right panel) overexpression diminishes NF-κB target gene expression. Infected HeLa cells were treated with DOX for 72 hr and stimulated with IL-1β for 60 min. Expression of indicated transcripts was analyzed by qRT-PCR. Bars show mean and standard error of the mean (SEM) of five independent experiments. (D) Schematic representation of YOD1 shRNA construct. GFP and shYOD1 were expressed under control of EF1α and H1 promoter, respectively. Both promoters are DOX/tTR-KRAB-controlled. (E) YOD1 protein levels are reduced in shYOD1 cells. Cells were treated for 72 hr with 0,05–0,5 µg/ml DOX as indicated and YOD1 knock-down was analyzed by Western Blot. (F) YOD1 knock-down results in enhanced NF-κB target gene expression. shYOD1-infected HeLa cells were treated with DOX for 72 hr and stimulated with IL-1β for the indicated time points. RNA was isolated and transcripts were analyzed by qRT-PCR as indicated. Bars show mean and SEM of four independent experiments. (G) TRAF6 and YOD1 exert opposing effects on NF-κB signaling and activation in iBMDM. iBMDM transduced with control shMock, shTRAF6 or shYOD1 were stimulated with IL-1β as indicated. NF-κB and Oct-1 (control) DNA binding was assessed by EMSA (n.s. = non-specific band). IκBα phosphorylation, degradation and knock-down efficiencies were analyzed by Western Blotting. (H) YOD1 knock-down promotes, while TRAF6 depletion impairs NF-κB target gene expression in iBMDM. iBMDM transduced as in (G) were stimulated with IL-1β for 45 min. Transcript levels were analyzed by qRT-PCR as indicated. Bars show mean and SEM of seven independent experiments. Significance was evaluated using Student’s t-test (*p<0,05; **p<0,01; ***p<0001; ns = not significant).