Fig 4.
Activation of RAW 264.7 cells treated with RNA polymerase III inhibitor ML60218. (A) RAW 264.7 cells pretreated with 20 μm of ML60218 (+) or DMSO (− [solvent control]) for 10 h were mock infected or infected with Ad5CiG virus (20,000 p/cell) and lysates harvested at 6 and 24 h postinfection. Protein lysates were analyzed by Western blotting as previously described. (B) RAW 264.7 cells pretreated with 20 μm of ML60218 (+) or DMSO (− [solvent control]) for 10 h were Lipofectamine 2000 transfected with increasing amounts of poly(dA-dT) or Ad5CiG DNA; lysates harvested at 5 h were analyzed by Western blotting as previously described. (C and D) RAW 264.7 cells treated as described for panel A were harvested for RNA at 6 h postinfection (C) or 24 h postinfection (D). qRT-PCR values for cellular control RNAs (Actin or M6tRNA), for virus-specific transcript (CAT or VAI), or for antiviral response-inducible transcripts (IFNB, ISG56, or TNF-α) were normalized to cellular HPRT as described in the text and Materials and Methods. The number above each bar indicates the unnormalized average CT value determined using biological triplicates for each transcript (see text).