Fig. 4. Intracellular calcium increase induced by PACAP-Epac2 signaling plays a key role in astrocytogenesis. (A) Wild-type (WT) neural precursor cells (NPCs) treated with PACAP in calcium-containing media showed prominent GFAP expression, whereas WT NPCs treated with PACAP in calcium-free media exhibited markedly lower astrocytic differentiation (trial number = 3). (B) Application of caffeine (100 μM or 1 mM) to Epac2-knockout (KO) NPCs raised the intracellular calcium concentration (trial number = 3). (C) Caffeine-induced increase in intracellular calcium levels by release from intracellular calcium stores recovered PACAP-induced astrocytogenesis in Epac2-KO NPCs in a dose-dependent manner, as compared to vehicle-treated KO NPCs (trial number = 3). Scale bars = 200 μm for A and C; Scale bar = 25 μm for B.
