For reproducing the anoxic environment, we used a hypoxic/anaerobic chamber (BBLTM GasPakTM, Franklin Lakes, NJ, USA). The system was set up at 37 °C in 5% CO2, 95% N2. Cells were transferred into the humidified chamber and incubated with the appropriate media for 24 h. The cells were then detached and analyzed according to the experimental plan. Control cells were incubated under normoxic conditions.
Arecaidine Propargyl Ester hydrobromide (Ape, Sigma-Aldrich, Milan, Italy) is a synthetic alkaloid obtained from modification of areca nut arecaidine. Its ability to selectively bind M2 muscarinic subtype has been largely demonstrated by pharmacological binding and M2 knockdown experiments [17 (link),18 (link)]. Cells were treated with 100 µM Ape, considering that this concentration was able to negatively control cell growth both in GBM cell lines and in GSCs, as previously demonstrated [18 (link)].
Arecaidine Propargyl Ester hydrobromide (Ape, Sigma-Aldrich, Milan, Italy) is a synthetic alkaloid obtained from modification of areca nut arecaidine. Its ability to selectively bind M2 muscarinic subtype has been largely demonstrated by pharmacological binding and M2 knockdown experiments [17 (link),18 (link)]. Cells were treated with 100 µM Ape, considering that this concentration was able to negatively control cell growth both in GBM cell lines and in GSCs, as previously demonstrated [18 (link)].
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