Expression of JAZ-GFP induces G₁ cell-cycle arrest followed by apoptosis in a p53-dependent manner. JAZ-GFP/pEGFPN1 was transfected into p53^+/+ (A) and p53^–/– (B) MEFs and NIH3T3 cells (C). pEGFPN1 (GFP) alone was used as a control. At 24, 48, and 72 hours following transfection, cells were harvested, fixed by 1% formaldehyde at 4°C for 1 hour, permeabilized by 70% ethanol at 4°C overnight, and stained by propidium iodide followed by flow cytometry analysis as described.⁵⁰  To normalize for transfection efficiency, only JAZ-GFP– or GFP-only–expressing cells were gated and then analyzed for a sub-G₁ population and cell-cycle distribution. According to the program used here, the sub-G₁ population represents the percentage of total gated (GFP or GFP positive) cells that are apoptotic, while the sum of the percentages of G₁/G₀, S, and G₂/M is normalized to 100%. This is done in order to accurately compare the cell-cycle distribution of the viable cells expressing JAZ-GFP or GFP-only. The arrows indicate sub-G₁ populations.