Fig. 11
Inhibition of neurogenesis but enhancement of astrogliogenesis in siATP6AP2 cells and depletion of SM/CL failed to recover ATP6Ap2. a, b hADSCs derived from healthy subjects transfected with an ATP6AP2-targeting siRNA (siATP6AP2) for 48 h (day –2/0) and then differentiated with 1% FBS. a Evaluation of ATP6AP2 mRNA and CLR-M protein levels as well as several key factors during differentiation of ATP6AP2-knockdown cells from day < 0 to day 37: neuronal TUJ1 and astrocyte GFAP markers; CAV and FLOT proteins in CLR-Ms; Gαi and Gαq proteins in CLR-Ms; phospho-ERK1/2/CREB/c-Jun/JNK proteins. Obtained cells at day 37 treated with hRenin (50 nM) for indicated time course to evaluate renin responsiveness. siATP6AP2 cells also treated with Gαq protein inhibitor YM254890 (YM) or Gαi inhibitor pertussis toxin (PTX) as indicated in Fig. 10 to determine their impacts on exosome release and intracellular calcium concentration. b Impact of CLR-M disruption during siATP6AP2-hADSC differentiation derived from healthy subjects: at day 0, cells were treated with or without nSMase (1 mU/ml) and/or MBCD (10 nM) for 48 h. c Impact of nSMase on hADSCs derived from ND patients during Neu-Dif: ATPAP2 and caveolin proteins in CLR-Ms, expression of neuronal marker TUJ1, Gαi and Gαq proteins in CLR-Ms, phospho-ERK/JNK, and exosome release. *P < 0.05,**P < 0.01: siATP6AP2 vs control. §P < 0.01: nSMase/MBCD vs control. ADSC adipose-derived mesenchymal stem cell, CAV caveolin, CLR-M caveolae/lipid raft plasma membrane microdomain, CREB cAMP response element-binding protein, D day, ERK extracellular signal-regulated kinase, FLOT flotillin, hRenin human recombinant renin, JNK Jun N-terminal kinase, MBCD methyl-β-cyclodextrin, ND neurodegenerative disease