Journal: BMB Reports
Article Title: Epac2 contributes to PACAP-induced astrocytic differentiation through calcium ion influx in neural precursor cells
Figure Lengend Snippet: Epac2 is involved in PACAP-induced astrocytogenesis. (A) GFAP immunolabeling of neural precursor cells (NPCs) at day 0 (DAT0). NPCs from neither wild-type (WT) nor Epac2-knockout (KO) mice expressed GFAP at DAT0. (B) WT NPCs remarkably differentiated into astrocytes when cultured in bFGF-free media for 4 days (at DAT4), but KO NPCs showed much lower GFAP immunoreactivity than WT NPCs. (C) CNTF (50 ng/ml) induced differentiation of NPCs of both genotypes into astrocytes. (D) WT NPCs incubated in PACAP (100 nM)-treated media for 4 days expressed GFAP, but KO NPCs did not display astrocytic differentiation. (E) Ratio of GFAP-immunopositive cell number to total NPCs number. KO NPCs did not show GFAP-immunopositive astrocytes by DAT4 in bFGF-free media ( # P = 0.0001, independent-samples t -test, trial number = 8). Treatment with either CNTF or PACAP increased differentiation of WT NPCs into astrocytes compared to WT NPCs without treatment at DAT4 (*P = 0.003 for CNTF, *P = 0.002 for PACAP, independent-samples t -test, trial number in each experiment = 8), but KO NPCs did not induce astrocytic differentiation after PACAP treatment contrast to CNTF treatment ( ## P = 0.0002, independent-samples t -test, trial number = 8). (F) GFAP intensity of immunopositive cells. KO NPCs showed much lower intensity in GFAP immunoreactivity compared to WT NPCs at DAT4 in bFGF-free media ( # P = 0.0066, independent-samples t -test). Treatment with either CNTF or PACAP increased GFAP intensity in WT NPCs compared to WT NPCs without treatment at DAT4 (*P = 0.024 for CNTF, *P = 0.007 for PACAP, independent-samples t -test), but contrast to CNTF, KO NPCs did not increase GFAP immunoreactivity even after PACAP treatment compared to WT NPCs ( ## P = 0.00004, independent-samples t -test). Scale bar = 200 μm.
Article Snippet: For astrocyte differentiation, cells were cultured in DMEM with N2 or calcium-free DMEM (Gibco, Thermo Fisher, Waltham, MA) with N2, which was then replaced with bFGF-free DMEM with N2 and a differentiation factor, such as 100 nM PACAP (A1439, Sigma- Aldrich, St Louis, MO) or 50 ng/ml CNTF (1203158, PeproTech) for 4 days.
Techniques: Immunolabeling, Knock-Out, Mouse Assay, Cell Culture, Incubation