Fig. 3. Effect of incubation temperature on the amount of HCMV IE transcripts after RNA polymerase II inhibition by DRB. (A) Integrated fluorescent intensity of HCMV IE transcripts accumulated in the nuclear foci relative to the integration site. The mRNA amount in the nuclear foci remained relatively constant during incubation of cells with DRB at 4°C. Shifting cells back to 37°C restored the previously observed decrease in integrated fluorescence intensity (half time 2 minutes). (B) Integrated fluorescent intensity of HCMV IE transcripts dispersed through the nucleoplasm relative to negative nuclei. The mRNA amount in the remainder of the nucleoplasm remained relatively constant during incubation of cells with DRB at 4°C. Shifting cells back to 37°C restored the previously observed decrease in integrated fluorescence intensity (half time 10 minutes). (C) CSLM images of cells at different incubation temperatures during intervals of DRB treatment. After induction of the HCMV IE gene expression by cycloheximide at 37°C, transcripts were observed in nuclear foci and in the remainder of the nucleoplasm. These transcripts remained present when incubation with DRB was carried out at 4°C. Subsequent incubation at 37°C resulted in a decrease in integrated fluorescence intensities with similar kinetics to cells treated with DRB at 37°C only.