I treated MDA-MB-468 cells with 10ug/ml ActD and collected samples with 2 hours intervals for 36 hours. However, my qRT-PCR result was not reasonable and I thought that i need to check if my treatment worked. so I need to find a gene whose expression definitely changes after ActD treatment in 468 cells. so can you recommend any gene or different way of controlling my treatment?
Hi there, I think the most straightforward approach might be to probe for activated/cleaved caspase-3 (WB analysis): it should go up significantly after several hours of treatment. At the same time, you can try WB and RT for Cyclin A and Cyclin D1 - both go down significantly on protein and mRNA levels. The concentration of ActD you are using is relatively high, so I would expect to see general inhibition of transcription in this case.
Good luck.
Actynomycin D should target DNA. So, you could cheque RT of genes coding DNA repair proteins and their phosphorylation using anti-phosphorylated mAbs (WB). It could be DNA protein kinase catalytic site (DNA-PKcs).
Best
I found two articles showing that c-myc and cyclin E decreases after ActD treatment (5ug/ml as far as I remember). They've studied different cell lines not MDA-468 but still these two genes can enlighten my way. Thank you so much for your valuable answers.
Regards
So Ayça, what is the final conclusion regarding the type of control to check actinomycin D treatment? Could you please inform me, thanks in advance.
Best regards,
İbrahim
Hi Ayça, What is the latest update about your treatment? What controls did you use? Thank you!
Hi Harun and İbrahim. I used c-myc for treatment control and it worked well. It showed decrease in first couple of hours after ActD treatment in MDA-231 and MDA-468 cells.
Dear Ayca, did you detect any difference at later time intervals or the most pronounced at 1-2 hours?
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