Sed protein-protein interactions among CXCR7 and CXCR4 upon CXCL12 treatment. In addition, to test if transfected CXCR7-mcherry or CXCR4-EGFP colocalized with endogenous proteins, we transfected hNPCs with either CXCR7-mcherry or CXCR4-EGFP and utilized immunocytochemistry to decide the endogenous CXCR7 or CXCR4 levels. Cells transfected with CXCR7-mcherry had been immunostained with endogenous CXCR4, and cells transfected with CXCR4-GFP had been immunostained with endogenous CXCR7. The confocal imaging showed immediately after CXCL12 treatment, CXCR7-mcherry and CXCR4-EGFP colocalized with endogenous CXCR4 and CXCR7, respectively (Fig. S6C). CXCL12 mediates hNPC survival via the ERK1/2 signaling pathway Activation of ERK1/2 by CXCL12 promotes cell survival on Retinal Ganglion cells 20. Moreover, current information has suggested that CXCR7 recruits -arrestin2 and increase ERK1/2 phosphorylation in cytoplasmic vesicles in HEK293 cells 21. To additional investigate the molecular mechanisms by which the CXCL12-induced endocytosis promotes cell survival, we tested no matter whether CXCL12 mediates hNPC survival via ERK1/2 signaling pathway. We pretreated hNPCs with ERK1/2 inhibitor PD98059 (20 M). Inhibition of ERK1/2 considerably lowered the anti-apoptotic impact of CXCL12 on hNPCs (Fig. 6A, B), Nav1.1 custom synthesis suggesting a essential function of ERK1/2 in CXCL12-mediated hNPC survival. To figure out whether or not endocytotic signaling is involved in ERK1/2 activation, we pretreated hNPCs with MDC (10 M) for 1 hour and determined ERK1/2 phosphorylation upon CXCL12 treatment through Western blotting. Quantification of Western blotting suggested that MDC blocks ERK1/2 activation at the time points amongst 5 and 60 minutes but not at earlier time points (Fig. 6C, D). This result is consistent with a prior report that showed the CXCL12-mediated ERK1/2 activation at early time points is mainly by means of GPCR signaling and the sustained activation of ERK1/2 at later time points is by way of endocytotic signaling 22, 23. To additional test regardless of whether ERK1/2 activation is dependent on CXCR7- or CXCR4-mediated endocytotic signaling, we transfected hNPCs with specific siRNA targeting either CXCR7 or CXCR4, and determined CXCL12-induced ERK1/2 activation by means of Western blotting. CXCL12 induced ERK1/2 phosphorylation in hNPCs. CXCR7 silencing considerably reduced CXCL12-mediated ERK1/2 phosphorylation at later time points (5 minutes to 60 minutes) (Fig. 6E, F). In contrast, CXCR4 silencing blocked both12-LOX Inhibitor Formulation NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptStem Cells. Author manuscript; offered in PMC 2014 March 29.Zhu et al.Pageearly (2 minutes) and late (5 minutes to 15 minutes) ERK1/2 activation (Fig 6G, H). These data suggest that sustained ERK1/2 activation by CXCL12 is dependent on each CXCR4 and CXCR7. Additionally, hNPCs were transfected with CXCR7-mcherry and treated with CXCL12 (one hundred ng/ml) for 30 minutes. Then we detected the ERK1/2 activation in hNPCs by immunocytochemistry. The confocal imaging showed just after therapy with CXCL12 for 30 minutes, the ERK1/2 have been activated at CXCR7-positive endosomes (Fig. S7). Taken together, we demonstrated that CXCL12 enhances hNPC survival by means of CXCR7- and CXCR4-mediated endocytotic signaling and ERK1/2 activation. Elevated apoptosis of NPCs in the developing brain of CXCR7 deficient mice To additional decide the relevance of CXCR7 in NPC survival in vivo, we obtained an established CXCR7 knockout mouse model 24. The migration defects of inter.