Figure 1.

Reduction of RNA polymerase II-mediated transcription by sublethal concentrations of α-amanitin and actinomycin D. (A) Experimental design scheme. (B) [¹⁴C]Thymidine-prelabeled NIH3T3 cells were treated for 16 h with 0–5 μg/ml of α-amanitin and pulse-labeled with [³H]uridine for 60 min at the indicated time points. Transcription rates were calculated on a per DNA basis as described in Materials and methods. Pretreatment of NIH3T3 cells with 3 μg/ml of α-amanitin for 16 h reduced RNA synthesis to 58±13% relative to untreated control (n=5); with 5 μg/ml of α-amanitin to 36±6% (n=4; panel 0 h). At 24 h after α-amanitin removal, the relative transcription rates amounted to 42±14% (n=5) and 26±6% (n=4), respectively, for the two drug concentrations (panel 24 h), and 48 h after drug removal, they amounted to 81±24% (n=5) and 29±5% (n=4; panel 48 h), respectively. (C) Relative run-on transcription rates were determined for nuclei of NIH3T3 fibroblasts pretreated with 3 μg/ml of α-amanitin for 16 h or with 50 nM actinomycin D for 5 h. (D) Expression profiles of c-fos precursor RNA from untreated cells and cells pretreated with 3 μg/ml α-amanitin during 16 h. Cells were collected at the indicated time points after horse serum addition (time 0); transcripts were quantified as described in Materials and methods. (E) Western blot analysis of whole-cell extracts from untreated cells and cells pretreated with 3 μg/ml α-amanitin. The antibodies used recognize the RPB1 and the splicing factor U2AF65 (loading control).