Fig 2: SAMPs activate the TLR8/MyD88/NF-?B axis in moDCs.(A) HEK293 cells stably transfected with human TLR7, TLR8, or luciferase alone (-) were stimulated with SCV2-RNA for 24 hours. NF-?B activation was evaluated as luciferase activity. Data are expressed as mean ± SEM (n = 3); *P < 0.05 versus (-) by 1-way ANOVA with Dunnett’s post hoc test. (B–E) moDCs were stimulated with SCV2-RNA as indicated (B) or pretreated with CU-CPT9a (1 µM) and then stimulated with SCV2-RNA or LPS for 1 hour (E). Nuclear extracts were blotted against NF-?B p65 and lamin B. One representative donor and densitometry of 3 donors are shown. *P < 0.05 versus untreated by 1-way ANOVA with Dunnett’s post hoc test; #P < 0.05 versus “SCV2-RNA” by paired Student’s t test. (C, left panel) moDCs were transfected with indicated siRNAs and target gene expression evaluated by qPCR. Results depict percentage of target gene expression (mean ± SEM n = 4). (C, right panel) moDCs transfected with indicated siRNAs were stimulated with SCV2-RNA for 24 hours and IL-6 production evaluated by ELISA. Data are expressed as percentage of production (n = 4); *P < 0.05 versus “ctr siRNA” by 1-way ANOVA with Dunnett’s post hoc test. (D) moDCs were transfected with indicated siRNAs and the expression of TLR8 was evaluated by qPCR (left y axis, white bars). IL-6 production upon SCV2-RNA stimulation was evaluated by ELISA (right y axis, gray bars). Data (percentage of expression/production) represent the mean ± SEM (n = 3); *P < 0.05 versus respective “ctr” by paired Student’s t test. (F) moDCs were pretreated with CU-CPT9a, then stimulated as indicated for 24 hours and IL-6 production evaluated by ELISA. Data are expressed as percentage of production for each individual stimulation (n = 3); *P < 0.05 versus respective “0” by 1-way ANOVA with Dunnett’s post hoc test.