In one of the cell line, the knockdown of my target gene (SATB1) shows an upregulated transcription of Cyclin B1 and D1 (q-RT-PCR). However, cell cycle analysis (flow cytometry) by Nocodazol block shows reduction in the rate of cell cycle, that is less number of cells reach G2/M phase at a given time point (12 h block of nocodazol).
So the results of flow cytometry are contradictory to the results of q-RT-PCR.
Any suggestions, comments are appreciated.
Thank you.
Hey Omkar,
Up-regulated transcription does not necessarily mean that the proteins are synthesized. It's possible that the knockdown of SATB1 in this cell line lead to some block in their cell cycle already and the increased transcription of cyclin D1 and B1 might be some compensatory mechanism.
Is there a chance these cells are having troubles in an earlier stage of the cell cycle e.g. DNA replication phase (S phase)?
Do you have a control group that was not blocked by Nocodazole (only SATB1 knockdown)? Are the cells there with increased cell cycle progression, more cells in G2/M? Is their cell growth correlating with the increased cyclin D1 and B1?
Have you checked other cyclins ( e.g. E and A), or CDKs?
I think it'll be good if you could check actual levels of the proteins by western blot.
Good Luck
Yosif
HI Yosif
Thank you very much for your input.
I did not check cyclin E and A or CDKs. I did not check the cell cycle rate without applying nocodazol block.
However, there is a strong anti proliferation upon SATB1 knockdown. Also Caspase 3/7 activity is increased upon SATB1 knockdown.
I will search for other markers.
Thank you very much once again!
Best,
Omkar
Hi Omkar
I agree with Yosif and also as suggested by Yosif you should check the protein level by WB analysis
Best wishes...
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