We have determined that our gene overexpression in dermal fibroblasts resulted in decreased cell proliferation and migration. Western blotting results showed that this effect is due to inhibition of PTEN/AKT pathway. To confirm these results, we want to use PI3K inhibitor LY294002, and PTEN inhibitors (bpv(HOpic), bpv(pic) and SF1670). However, I have found that there is not an optimized protocol for the incubation time and concentration. Should I treat cells for 30 min, or 72h to detect cell proliferation. Or, during 72h treatment, should I change medium containing inhibitors every day?
Secondly, LY294002 are claimed to respress cell viability (measured by WST1 or MTS) in all articles I have read. How should I confirm that this reduction is not arisen from cell toxicity of LY294002, what would be the best method to confirm this? I appreciate any thoughts and suggestions.
Thanks in advance
Generally speaking, small molecular inhibitors become inactive in serum-containing medium after 12-24hrs, so that I strongly recommend you should replace the medium with the inhibitor of interest every 12-24hr. Only after these replacements, you can exactly assess the inhibitory effect.
i agree with yoshida,
perhaps you should first look for proliferation and apoptosis by using for detection of the right/most efficient concentration of your inhibitors.
i work with LY too in vitro and before i adjusted the concentrations, i performed MTT and PI. perhaps you can look for the IC50 of your inhibitiors.
and it depends on which targets your are interested in. cell signaling or other things. if your interested in mechanistical stuff including signaling, you can use shorter time periods of incubation.
For the measurement of proliferation I would use different time points: 24, 48, 72, 96 h. And in parallel, I would control for apoptosis (Annexin V / PI staining). Since PI3K is one of the classical anti-apoptotic pathways, it is very likely (almost normal) that after a certain time PI3K inhibitors cause a reduction in cell viability. Maybe you can find a time point, at which proliferation and migration are affected by LY treatment and the cells still are viable.
Moreover, LY294002 is not the best PI3K inhibitor since, amongst hoers, it also inhibits the Pim kinases very well and these kinases also act in an anti-apoptotic fashion. It would be better to use a more selective PI3K inhibitor such as PI-103.
Furthermore, are you sure that you want to inhibit PTEN? PTEN hydrolyzes the product of the PI3K-catalyzed reaction, PIP3, and inhibiting it would even enhance the effects of PI3K.
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