Figure 6

a, The synthetic dsDNA template AT+30N or AT70 was transfected into 293T cells, whereas non transfected cells were used as a control. 8 h after transfection total RNA was isolated, polyadenylated and reverse transcribed as depicted in supplementary Fig. 6. The obtained cDNA was then used for PCR using a specific upstream primer for either 5S rRNA or the 30N transcript and a downstream primer binding within the RT primer. A standard PCR was done with primer pairs for β2 microglobulin. A representative PAGE analysis is shown for a conventional PCR amplification for all three primer combinations. In addition a real-time PCR analysis is shown for the AT+30N transcript normalized to β2 microglobulin expression. b, 293T cells were transfected with (AT)35, AT+30N, AT+6T+30N dsDNA or left untreated. RNA was isolated and treated as above and 3′ RACE real time PCR was performed for the 30N tag. The 30N tag expression data were normalized to β2 microglobulin expression, c, 293T cells were treated with ML-60218 for 2h at the indicated concentrations and transfected with AT+30N dsDNA. 16h later RNA was isolated and the expression of the 30N tag normalized to β2 microglobulin expression was assessed. d, 293T cells were treated with ML-60218 for 2h at the indicated doses. Subsequently cells were transfected with an IFN-β reporter plasmid in conjunction with poly(dA-dT) or SEV. After an additional period of 24h transactivation of the IFN-β promoter was measured. In addition, 293T cells were reverse transfected with two individual siRNAs targeting the indicated genes. After 2 days cells were transfected with AT+30N and after an additional period of 16h transcription of 5S rRNA (e) and AT+30N (f) was determined via 3′ RACE PCR. g, 293T cells were reverse transfected with four individual siRNAs targeting the indicated genes. 48h after transfection cells were transfected with an IFN-β reporter plasmid in conjunction with poly(dA-dT) or SEV. After an additional period of 24h transactivation of the IFN-β promoter was measured. Four individual siRNAs were used per gene and were each tested in triplicates. Data are represented as mean values normalized to the set of control siRNAs. Mean values ± SEM of one representative experiment out of two (c, e, f), three (a, b, g) or four (d) experiments are depicted.