Panx1 expression in NSC/NPCs is re-capitulated in vitro in neurosphere cultures. (A) Outline of neurosphere culture generation from P0 to P3 hippocampus or microdissected VZ. Spheres are cultured for seven days in vitro (DIV) with addition of growth factors (hEGF and bFGF) every two DIV, before harvesting for subsequent analyses, or differentiation by removal of growth factors and plating on a poly-D-lysine coated surface. (B) Panx1 mRNA (i) and protein (ii) are expressed in VZ and SGZ neurospheres. To the left of the western blot, the asterisk (*) above the arrow denotes high-molecular weight species likely representing various Panx1 oligomeric forms, while the asterisk below the arrow denotes low molecular weight Panx1 cleavage products consistent in size with those reported in the literature . (C) Confocal images showing Panx1 expression in a cryosectioned undifferentiated VZ neurosphere (i) and a digitally zoomed z-stack with orthogonal side-views (ii) in nestin-positive/GFAP-positive and nestin-positive/GFAP-negative cells. (D) Image from a confocal z-stack with orthogonal side-views of Panx1 expression in DCX-negative cells in a differentiated VZ neurosphere. (E) Image from a confocal z-stack with orthogonal side-views showing high levels of Panx1 co-expression with Tuj-1 in immature neurons from a differentiated VZ neurosphere. Hoechst 33342 was used as a nuclear counterstain in C-E. Scalebars 10 μm. DCX, doublecortin; GFAP, glial fibrillary acidic protein; NPC, neural progenitor cells; NSC, neural stem cells; Panx1, pannexin1; SGZ, subgranular zone; VZ, ventricular zone.