I am working with iPSC-derived macrophages and looking at autophagy in these cells. Rapamycin induces expression of LC3II while bafilomycin prevents its turn over. From my data (and previous data in the field) addition of rapamycin causes a significant increase in LC3II levels, so what is the purpose of having rapamycin and bafilomycin treated cells? My understanding was that it enhances LC3II levels but if they are already significantly higher in rapamycin treated cells, is there a need to add bafilomycin?
Hello Priyadarshini Chatterjee
If the cells express high basal levels of autophagy, you may block autophagy using bafilomycin treatment that will induce cell death. You may treat the cells with different concentrations of bafilomycin in a dose/time dependent manner, with or without rapamycin. Bafilomycin induces cell death in concentration/time-dependent manner and rapamycin treatment will attenuate this bafilomycin effect.
So, to see the actual effect of rapamycin on autophagy, treating the cells with both, bafilomycin and rapamycin becomes essential as rapamycin will attenuate the bafilomycin effect by bringing about upregulation of autophagy in the cells that are treated with bafilomycin in a dose/time-dependent manner.
Best.
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