Panx1 levels decrease across neuronal differentiation and are important for neuritogenesis. (A) Confocal image of VZ-derived cells under neuronal driving conditions immunolabelled for endogenous Panx1 (left). Arrowheads indicate Panx1 in the neurite. VZ neurospheres were replated and maintained in proliferative conditions (UD; un-differentiated) or neuronal driving conditions (D; differentiated) for 5 days. Panx1 expression assessed by Western blotting (middle) was significantly lower in differentiated compared to undifferentiated neurospheres (right). (B) N2a cells were differentiated for 24 hours in low-serum media with 10 μM retinoic acid. Samples were collected at 0, 2, 6, and 24 hours. (Left) Confocal image of endogenous Panx1 staining at 24 hours of differentiation. Arrowheads indicate Panx1 in neurites. Western blotting (middle) revealed significantly reduced Panx1 expression in 24 hour differentiated samples compared to 0 hour controls (right). (C) Representative images of VZ NSC/NPCs (dissociated neurospheres) treated with 1 mM probenecid or vehicle control for 48 hours. A process with length greater than or equal to the corresponding cell body length was considered a neurite. (D) The percent of VZ NSC/NPCs possessing a neurite increased with probenecid treatment. (E) The length of VZ NSC/NPC neurites increased with probenecid treatment compared to control. (F) Probenecid treatment significantly increased the average number of neurites per cell in VZ NSC/NPCs compared to control, and (G) dramatically altered the neurite number distribution. (H) Representative images of N2a cells treated for 36 hours with 1 mM probenecid or vehicle control. (I) Probenecid treatment increased the percent of cells possessing a neurite. (J) Panx1 siRNA knockdown increased the proportion of cells possessing a neurite. (K) Representative image of N2a cells transfected with Panx1EGFP 24 hours after induction of differentiation. (L) Significantly fewer N2a cells overexpressing Panx1EGFP possessed one or more neurites compared to untransfected same-plate controls. Hoechst 33342 was used as a nuclear counterstain. All scalebars are 10 μm.