At 24 h from seeding, cells were incubated in the presence of the M2 muscarinic receptor agonist arecaidine propargyl ester (APE) (100 µM) (Sigma-Aldrich, St. Louis, MO, USA) for different time points according to the experimental plan (24 h, 48 h). The selective binding of APE to M2 receptors has been previously demonstrated by pharmacological competition binding assay and M2 silencing experiments both in the GBM cell lines and in GB cancer stem cells [18 (link),19 (link),34 (link)].
GBM cells were also incubated for 24 h with 5 µM N-[N-(3,5-difluorophenacetyl)-l -alanyl]-(S)-phenylglycine t-butyl ester (DAPT) (Sigma-Aldrich, St. Louis, MO, USA), a gamma-secretase inhibitor or 10 µM N-(3-Chlorophenyl)-6,7-dimethoxy-4-quinazolinamine (Tyrphostin; Tyrph) (Sigma–Aldrich, St. Louis, MO, USA), as an inhibitor of EGFR activity.
GBM cells were also incubated for 24 h with 5 µM N-[N-(3,5-difluorophenacetyl)-
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