Fig. 1. (A) Cell cycle profiles of asynchronised and synchronised COS-7 cells. Propidium Iodide (PI) was added to COS-7 cells so that their DNA content could be measured by flow cytometry, and thus the stages of the cell-cycle identified. The black line shows the cell-cycle profile for asynchronised COS-7 cells. The cells are in G1, S and G2M phase, although the majority of the cells are in G1. Cells were synchronised by nocodazole treatment; the blue line shows that now at least 50% of cells are arrested in G2M. (B) Flow cytometry can be used to study EGFP-tagged proteins during the cell cycle. COS-7 cells were transfected with EGFP-emerin constructs and examined by flow cytometry. The untransfected population (dotted black line box) and transfected population (solid black line box) could be gated to allow each population to be analysed separately. The gates also allowed doublet cells or cells expressing a high amount of EGFP to be excluded from the analysis. (C) Cell-cycle profiles of untransfected and EGFP-emerin-transfected cell populations. The PI area (DNA content) and EGFP intensity were both monitored so that cell-cycle profiles show the relative number of cells in G1, S or G2M for each population (untransfected and transfected). In each profile, the orange lines represent the untransfected populations and the green and red lines represent cells expressing EGFP-emerin 12 and 24 hours after being released from G2M. The untransfected population acts as an internal control to compare the length of the cell cycle with that for cells transfected with either wild-type or mutant forms of emerin. The cells expressing the emerin mutant Del236-241 are shown to have a prolonged cell cycle length.
