Figure 4. RING105 expression delays the G1-to-S transition in HeLa cells.

(a) RING105 transfection to cycling HeLa cells. HeLa cells were transfected with plasmids expressing RING105-GFP constructs (see Figure 1b) and the effect on the cell cycle was analysed by flow cytometry 24–40 h after transfection. Experiments were repeated at least twice and typical results from GFP-positive cells are shown. Numbers indicate estimated G0/G1 percentage. Cells transfected with plasmids expressing full-length (+1) or the C-terminal region containing the RING-finger domain (+4) showed a substantial accumulation of G1 cells. Point mutations of the RING-finger domain in full-length TSSC5 (+2, +3) or in the C-terminal construct (+5, +6) significantly reduced accumulation of cells in G1. (b) RING105 transfection and DNA-damaging agent VM26 (teniposide) treatment. Transfected HeLa cells were treated with the topoisomerase II inhibitor VM26 (0.2 µg/ml, 12 h) leading to DNA damage. This treatment led to an accumulation of cells in S phase/G2 phase. In cells transfected with plasmids expressing full-length (+1.Full) and C-terminus RING-finger domain (+4.C) of RING105, the S/G2 peaks were reduced, suggesting that the transfection causes delay in the cell cycle progression before S phase. (c) RING105 transfection and spindle inhibitor nocodazole treatment. HeLa cells were transfected with empty plasmid (vector) or with plasmids encoding full-length RING105 (+1.Full), N-terminus (+7.N) and C-terminus (+4.C) of RING105 in the absence (left column) or presence (right column) of nocodazole (100 ng/ml, 16 h). Nocodazole induces mitotic-checkpoint-dependent G2/M arrest. Populations transfected with empty vector or with plasmids expressing the N-terminus showed the normal increase in the G2/M peak. Populations expressing the full-length RING105-GFP (+1) showed a diminished degree of accumulation, while cells expressing the C-terminus RING-finger domain (+4.C) showed almost no accumulation in G2/M (arrows).