In order to investigate the function of RNF8 in cell growth, two siRNAs termed siRNF8-1 and siRNF8-2 were transfected to HeLa
dapt secretase and cell numbers were counted for 4 days. Cell growth was inhibited in RNF8 siRNA-transfected cells (Fig. 1A). To clarify whether these cells were dead or just arrested, flowcytometrical analyses were performed. The RNF8 siRNA transfected cells were pulse labeled with BrdU and its incorporation was studied with anti-BrdU antibody. The percentages of S phase without DNA replication in siRNF8-1, siRNF8-2, or siGL3 at 72 h were 11.8, 12.8, or 2.4, respectively. These results suggest that RNF8 siRNA transfected cells were arrested at the S phase without BrdU incorporation (non-replicating). At the same time point, the percentage of cells at the G2/M phase of siRNF8-1 or siRNF8-2 transfected cells was high (150% or 163%) compared with control. These results indicate that RNF8 siRNA transfected cells were also arrested at G2/M phase. In addition, a part of the cells showed a lower intensity of propidium iodide staining, indicating that RNF8 siRNA caused cell death. Control GL3 siRNA transfected cells showed normal distribution of
cell cycle stages (Fig. 1B). Under this condition, the lower RNF8 protein content was confirmed in RNF8 siRNA-transfected cells compared with control siRNA-transfected cells (Fig. 1C). These results suggest that RNF8 has functions in S phase and G2/M phase progression and in the checkpoint mechanism.
