Cpg2006

RNA interference was performed by lipofection (RNAiMax, Life Technologies) of 5 nM Silencer Select siRNAs (Life Technologies) for 72 h. Cells were stimulated for 14 h with CpG2006 (Metabion), R848 (InvivoGen), CL075 (InvivoGen), Pam2CSK4 (EMC microcollections), human TNF or IL-1β (R&D systems), TLR7-specific RNA (5′-ACUG1CG1AG1CUU-X-UUCG1AG1CG1UCA-5, G1 is 7-deazaguanosine, X is 1,2,3-propanetriol) (14 (link)) or TLR8-specific RNA (5′-YUGCUGCCUUUG-X-GUUUCCGUCGUY-5′, Y is 1,3-propanediol, X is 1,2,3-propanetriol) (15 (link)) (Idera Pharmaceuticals). Supernatants were analyzed by ELISA for hIL-8 (BD Biosciences), hIL-6 or hTNF (R&D Systems). Efficiency of RNA interference was analyzed by SYBR Green quantitative PCR (qPCR) for MyD88, AP1M1 and AP2M1 expression normalized to HPRT.

Cells were stimulated as indicated with TLR7-stimulatory RNA (5’-ACUG1CG1AG1CUU-X-UUCG1AG1CG1UCA-5’, G1 is 7-deazaguanosine, X is 1,2,3-propanetriol) from Idera Pharmaceuticals, R848 (InvivoGen), TLR13-stimulatory RNA (ORN Sa19, InvivoGen), LPS (InvivoGen), CpG1826 PTO (Metabion), CpG2006 (Metabion), heat-killed GBS (kind gift from Dr. G. Teti), human TNF (R&D Systems) or human IL-1β (R&D Systems). Transient transfections were performed using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific). Where indicated, cells were pre-incubated with Bafilomycin A1 (biomol). Cytokines were measured using commercially available ELISA kits (R&D Systems and BD Biosciences).