Hello Konstantin, I have two question to make you. What concentration of doxorubicin are you using? how long do you wait to see the phosphorylation of H2AX? I have used 10 nM of doxorubicin in MDA-MB-231 cells and that was enough to see phosphorylation of H2AX for 30 minute. You probably alredy know this, but the maximus phosphorylation of H2AX is during the first 30 minute of treament.

Hi Konstantin Byrgazov , Very interesting results...!

Just a couple of things before hand (may be you know this already)

1. H2AX can be phosphorylated by two major DNA damage response (DDR) kinases: ATM (in response to double strand breaks) and ATR (in response to replication stress or single strand breaks (SSBs)). Although H2AX phosphorylation is widely used as a proxy for double strand breaks (DSBs), it can be possible that this event occurs in response to SSBs or replication stress. So, may be you want to check the activation profiles of ATM and ATR in your experiments.

References: "H2AX Phosphorylation: Its Role in DNA Damage Response and Cancer Therapy"

2. Chk2 can be activated in both ATM-dependent as well as ATM-independent manner. From your results, it seems to me a possibility that chemicals used in the study (at their respective concentrations and timings) might activate signal transduction pathways differently.

References: "Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner."

Thanks

Satish