No more apoptosis, no less proliferation but less cells... - (Feb/03/2007 )
I'm carring out some experiments using siRNA for silencing a protein. I see that when my protein is knock-down, there are less cells (when I count them) in comparison to scrambled treated cells. The problem is that I thought there would be two possibilities:
1. The protein is implicated in proliferation so when I reduce it, cells stop growth.
2. The protein is implicated in apoptosis so when I reduce it, cells die.
Surprisingly, when I analyze cell cycle with a cytometer, I see that there is not a significant SubG0 population (no apoptosis) and neither a significant change in G1, S or G2/M populations.
So my question is: if I see fewer cells and they dont growth less and not die more, what is happening?
Thank you very much for reading!!
necrosis? Cell just pop open. Maybe protein is important in cell membrane integrity or osmotic and ionic balance.
Can you do time laps to direct watch the cells? Maybe you can just monitor the cells from immediately after transformationi till a few days after using some kind of camera attachment to the microscope.
Do you see a reduction in cell numbers when you use a control siRNA?
I am wondering if there is any sort of interferon response leading to loss of cells.
Perneseblue, I also checked necrosis but the result was both scrambled-treated and siRNA-treated cells were 95% iodine propidium negative.. there wasnt a signficant necrosis process.... they were alive.... It's a pity but in my faculty there isnt time laps equipment.
Scolix, I transfect the same amount of RNA when I use scrambled and specific siRNA (100 pmol) so in case of INF response it would affect both equally, wouldnt it?
Thank you for your answers!!!.... I keep thinking a solution (what else can I do?)
Quite right. One would see a IFN response in both equally. But sometimes, some siRNA could initiate IFN better than others for unknown reasons. ( its something you might have to prove for publication)
But as you observed fewer cells in the treated ones, have you quantified it.
It is possible that the protein is needed for cell survival and loss of this protien is leading to cell loss. Could you try to overexpress this protein in a cell survival assay invitro.
Or if the protein is involved in cell growth, do you actually observe significant differences in cell numbers.