Using a MUT vs parent cell line, my colleagues and I have seen increased proliferation in MUT cells. To address this further, we have been synchronizing cells 48h in 0.1% serum (with 10mM sodium butyrate added for the last 24h, G1/S block) and then releasing them back to 10% FBS and monitoring cell cycle progress using PI. I'm having some difficulty interpreting the data, so thought I would check on here to see if those more versed in cell cycle analysis can help. Here is what we are seeing:

1. Unsynchronized WT cells actually have reduced % cells in G2/M phase (14% vs 17% in parent cells, so mild).

2. After 48h of synchronization, we now see MUT cells having increased % G2/M phase (11% vs 9% in parent cells).

3. When we release the synchronized cells and put them back into 10% FBS, 12hr/16/20h later the MUT cells all have increased %G2/M (10%/6%/3.5% vs 6%/3%/2% in parent cells) as well as increased increased % S at 12h/16h (12%/13% vs 6%/9% in parent cells).

These differences, while statistically significant, seem mild, but would the interpretation be that the MUT cells have increased entry into G2/M phase? I was expecting to see a huge defect in the majority of cells that are synchronized, but at 24h when the bulk of the cells are in S phase, I don't see a difference between MUT and parent cells. I'm not sure how to interpret this, but am wondering whether I should consider synchronizing the cells at a different point in the cell cycle to try to bring out what appears to be acceleration to G2/M phase.

Any help is greatly appreciated! This is my first time giving cell cycle analysis a go so I'm a bit of a newbie here :)