Fig. 10
Activities of Gα protein-sensitive CREB/C-Jun, exosome releases and intracellular calcium during neural differentiation of hADSCs. hADSCs isolated from healthy subjects and differentiated for 37 days as indicated in Methods; cells treated for 48 h (day –2 to day 0) with Gαs activator cholera toxin (CTX, 1 μg/ml) or Gαq protein inhibitor YM254890 (YM, 1 μM) or Gαi inhibitor pertussis toxin (PTX, 100 ng/ml). Analyses performed along differentiation (a) or at NLC stage (day 37) (b). Nuclear extracts separated by SDS-PAGE and immunoblotted with antibodies directed against total and p-CREB and c-Jun. Secreted exosomes collected and quantitated by measuring AChE activity. Involvement of intracellular Ca2+ assessed by treating differentiated NLCs at day 37 with BAPTA-AM (25 μM, 24 h before adding hRenin) or by measuring [Ca2+]i during Neu-Dif and using Fura2/AM labeling as described in Methods. Neu-Dif evaluated by TUJ1 protein expression. ATP6AP2 responsiveness to hRenin after CTX/PTX/YM treatments evaluated in NLCs at day 37. **P < 0.01: D0/14/30/37 vs day < 0. §P < 0.01: treatments vs control. AChE acetylcholinesterase CREB cAMP response element-binding protein, D day, NLC neuron-like cell