Figure 3: Cyclin F is involved in controlling the transcriptional activity of B-Myb.

(a) U2OS cells were transfected with control (UNC) or cyclin F siRNA. The cells were synchronized by a double thymidine block and released for 8.5 h, followed by IR treatment (6 Gy). Cells were harvested 0 (−), 2, 4 and 6 h after IR and processed for immunoblotting. (b) U2OS or Frt-Cyclin F siRNA-resistant cells were transfected with UNC or cyclin F siRNA. After 48 h, cells were treated with IR (6 Gy) and harvested 6 h later. RNA was extracted, cDNA generated and relative numbers of transcripts were analysed by quantitative PCR. The expression levels of transcripts were normalized to the housekeeping gene ubiquitin in all samples. The values were subsequently normalized to the IR-treated control (UNC transfected) sample. Error bars indicate s.e.m. (n=3). (c) Left panel: U2OS p53 dneg cells were transfected with control (UNC) or cyclin F siRNA with or without B-Myb#1/#2 siRNA. After 48 h, cells were irradiated (6 Gy), 2 h later treated with Nocodazole for 7 h and harvested for flow cytometry to detect mitotic cells using anti-H3S10-p. Error bars indicate s.e.m. (n=3). Right panel: immunoblot of U2OS p53 dneg cells depleted for cyclin F and/or B-Myb#1/#2 for 2 days. (d) U2OS cells were transfected with control (UNC) or cyclin F siRNA. The cells were synchronized by a double thymidine block and released for the indicated time points. The whole-cell lysates and chromatin fractions were analysed by immunoblotting. (e) U2OS cells were transfected with control (UNC), cyclin F or B-Myb siRNA. The cells were synchronized by a double thymidine block, released for 8 h and left untreated or treated with IR (6 Gy) followed by harvesting 3 h later. The whole-cell lysates were analysed by immunoblotting with phospho-B-Myb-T487, B-Myb, cyclin F and vinculin antibodies. The asterisk denotes an unspecific band.