Figure 1: Ubiquitome library screen identifies a role for cyclin F in the IR-induced G2 checkpoint.
From: Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control
(a) Schematic overview of the automated DNA damage checkpoint siRNA screen with an ubiquitome library. p53 dominant-negative U2OS (p53 dneg U2OS) cells were reverse transfected, incubated for 2 days, irradiated with 6 Gy of IR and 2 h later Nocodazole (Noco) was added for 8 h. The cells were fixed and stained with anti-H3S10-p antibody and Hoechst and analysed for genes regulating the G2 checkpoint. (b) 384-well-plate images from the screen showing control (UNC), CHK1, BRCA2 and cyclin F-depleted cells. (c) U2OS or Frt-Cyclin F siRNA-resistant cells were depleted for control (UNC) or cyclin F. Twenty-four hours after transfection, doxycyclin was added where indicated to express the siRNA-resistant version of cyclin F. After another 24 h, cells were irradiated with 6 Gy of IR, left for 2 h, treated with Nocodazole for 7 h and harvested for flow cytometry. (d) Quantitative analysis of the experiment described in c. Mitotic index is calculated as IR-Noco/Noco relative to the control (UNC). s.e.m. is calculated from three independent experiments. (e) Immunoblot showing cyclin F depletion and overexpression corresponding to the samples in c and d.
