a and b, Mutu III cells were treated with ascending doses of ML-60218 for 24h and IFN-α production was assessed by ELISA (a, left panel) and cell viability was assessed using Calcein AM staining (a, right panel), while EBER RNA and 5S rRNA transcription was measured by real-time PCR (b). c, The genomic locus that encodes for EBER-1 and 2 RNA, EBER-1 RNA, EBER-2 RNA was PCR-amplified and transfected into IFN-primed 293T cells (all 120ng or as indicated). PCR-amplified EGFP served as a negative control, whereas poly(dA-dT) was used as a positive control. Subsequently transactivation of the IFN-β promoter was measured after 24h. d, IFN-primed 293T cells transfected with the full length EBER RNA gene locus were treated with ascending doses of ML-60218 and after 24h EBER-1, EBER-2, 5S rRNA and Cyclophilin B transcription was determined. e, In addition, transactivation of the IFN-β promoter was assessed in IFN-primed 293T cells that had been transfected with either poly(dA-dT), the EBER RNA gene locus or RIG-I (all 120ng). Data were normalized to the positive control RIG-I. f, 293T cells were reverse transfected with two individual siRNAs targeting the indicated genes. 48h after siRNA transfection cells were transfected with an IFN-β reporter plasmid in conjunction with the EBER RNA gene locus, poly(dA-dT) (all 120ng) or SEV. After an additional period of 24h transactivation of the IFN-β promoter was measured. Two individual siRNAs were used per gene and were each tested in triplicates. Mean values ± SEM of one representative experiment out of two (b, d, f), three (a, e) or four (c) experiments are depicted.