FIG. 4.

Induction of innate immune responses in DCs after Min6/CVB uptake requires (viral) RNA within Min6 cells. A: DCs were pretreated with CytD for 30 min prior to stimulation with TLR ligands or Min6 cells. After 8 h, mRNA expression levels of RIG-I, Mda5, and PKR were determined using qPCR. B: DCs were pretreated with CQ for 30 min, stimulated as in A, and mRNA expression was determined as in A. C: Min6 cell preparations were exposed to a mixture of RNase A, RNase VI, and RNase I prior to addition to DC cultures as described. Expression of RIG-I, Mda5, and PKR in DCs was analyzed using qPCR 5 h after addition of Min6-cell preparations. D: DCs were co-cultured with Min6-cell preparations as described for panel A, and protein expression of RIG-I, Mda5, and PKR was analyzed by Western blotting 24 h after the start of co-culture. Data are representative of two (D) or the average of two (A–C) independent experiments. In these experiments, freeze-thawed cell populations were used. IC, poly (I:C); Med, medium unstimulated cells; M6/CVB, CVB-infected Min6 cells; M6/M, mock-infected Min6 cells; n.s., not significant; w/o RNase or w RNase, without or with RNase treatment of Min6 cell preparations prior to co-culture. *P < 0.05.