Posted 08 May 2017 - 11:42 PM
I treated ny cells with a drug for 24hrs and checked cell cycle analysis by observing G1, S and G2 phases with PI staining. I got an:
-decrease in G1 phase in my treatment group vs ctrl group
increase in S phase in my treatment group compared to ctrl group.
I thought my cells are showing signs of proliferation, until I read somewhere that entry into S phase can also lead to apoptosis.
By western, p21 abd p27 show reduction in treatment group vs ctrl.
I don't know what to make of my results.
Posted 10 May 2017 - 06:08 AM
A single PI staining experiment isn't going to be sufficient to determine what this drug is doing in terms of proliferation, apoptosis, and cell cycle progression. Was there an increase in the G2/M peak? Reduction of p21 and p27 does indicate the potential for more proliferation but you can't be certain that your cells are indeed cycling. Do you know what is the doubling time for these cells?
First, a basic proliferation assay (SRB, crystal violet, cyQuant) is a good way to determine if your drug is influencing growth. You don't need many cells or conditions and you'll have that answer within a week. That's going to be very informative as to your drug's growth effect. It might be that your drug blocks or slows completion of S phase, in which case you'll see loss or reduction in growth compared to your controls.
There are many potential assays to be done here. First, a longer time course with drug treatment, western blot for different cyclins (is cyclin B up?), annexin V/PI or caspase activity for death, BrdU/EdU incorporation for DNA synthesis (I'd do this after a double thymidine block), MTT or MTS for metabolic viability, etc.
Posted 10 May 2017 - 03:33 PM
Thank you very much. I have done MTT as well as cyquant and cells show increased proliferation compared to control. I will try checking for caspase activity.