Hi everyone.
I 've just tested two classic SIRT1 activators (resveratrol and SRT1720) in my HUVECs.
Both compounds were dissolved in DMSO, and the final concentration ranges were 0-20 microM (RV) and 0-2 microM (SRT1720). For all treatments, we used a final DMSO concentration of 0.07 % (v/v), which is much lower than the most common DMSO concentration used in literature. Treatment duration was 24 hours for all conditions.
I analyzed SIRT1 protein levels by Western blotting.
Surprisingly, I could not see any increase in either RV- or SRT1720-treated cells (several literature reports show SIRT1 activation for 10 microM RV and 1 microM SRT1720).
However, I detected a dose-dependent decrease in alive cells number for both activators, and a significant increase in dead cells, only for the highest SRT1720 concentration, as compared to cells treated with DMSO only. This suggested that the two compounds were actually added the medium.
Here I attach the results for RV treatment only, because what I got for SRT1720 is the very same result.
Any help is highly appreciated.
Thank You very much in advance
Stefano Falone
Hi Stefano.
1) DMSO seems to be OK. Just check what cell lines are used in other papers. If they used the same cell line, then your cell culture system may not be in the optimum condition at present. Dirty culture might give all activated signals. The higher concentration above the threshold levels used further gave increasing rate of cell death.
2) The other suggestion is setting a trial pilot expt. to seek IC50 concentrations of the 2 activators to evaluate cell survival and death with PI and Hoechst together. Because the batches obtained and the way prepared are all different in the labs. This is really crucial in the subsequent expt.
Regards.
Thanks for Your kind answer, Prof. Sang Ho Lee.
I am using a pool of primary HUVECs from healthy subjects. I am not experiencing any problem with cell growth, and my HUVECs seem to adhere normally to flasks. Therefore, I am quite confident that the culture is not contaminated.
RV and SRT1720 were purchased from Sigma-Aldrich and S.I.A.L., respectively. The fact that cell growth was slowed by both compound in a concentration-dependent fashion shows that the compounds were actually added to the medium. Rather, I was thinking that maybe the treatment duration should be reduced. A time-course experiment would show a possible early effect that, supposedly, was lost by performing only a 24-hour incubation. In other words, maybe SIRT1 was actually activated by treatments with RV and/or SRT1720, but by detaching cells after 24 hours, this effect was not evident due to the restitutio ad integrum.
Right now, I am repeating the first experiment, just to be sure that what I found was not dependent on technical errors. If the result is the same, I will try a time-course experiment (e.g., incubation durations from 3 to 24 hours, with steps of 3 hours).
Thanks so much for Your precious and kind advice.
Regards
Stefano
I can't speak directly for your SIRT1 activators, but might your serum be interfering? I don't know what media formulation or serum levels you are using, but often HUVEC cells are cultured with high serum levels (up to 20% or so). However, low serum formulations (2% in some HUVEC Media formulations) may be more desirable for many treatments as serum is often considered to interfere with various treatments in many cell assays.
Good Luck, I hope this helps.
Dear Floyd,
I am treating my HUVECs in medium containing 2% serum, therefore I don't think that's the reason I don't see SRT1720-dependent effect (indeed, further experiments showed that 24-h 5 uM RSV was enough to increase SIRT1 expression).
Thanks anyway and best regards
Stefano
Yes, sounds like high serum levels were not the issue here.
Glad things worked out.
- Badges
- Science method