Figure 3. Neural precursor cell proliferation on endothelial cells is inhibited by ephrinB2 ligands.
(a) Quantitative RT-PCR analysis of mRNA levels of the indicated cell-cycle regulators in NPC cultured on their own (−) or together with endothelial cells (+bEND) in growth media for 24h (n=3 RNA extracts from independent experiments). Error bars denote s.d. Two-tailed paired Student’s t-test. (b) Western blot analysis of p-ERK and total ERK levels in NPC cultured alone (−) or with endothelial cells (+bEND) for 24h under the same conditions. (c) Top: Representative immunofluorescence images of cell-tracker labelled (green) NPC in monoculture or co-culture with endothelial cells (unlabelled) and pure endothelial cell control cultures stained for p-Eph (red). Nuclei were counterstained with DAPI (blue). Bottom: western analysis of pEph levels in NPC cultured alone (−), on bEND or ephrinB2-Fc for 24h, β-tubulin is used as loading control. (d) Immunofluorescence staining for N-cadherin (green) of NPC seeded on clustered ephrinB2-Fc ligands or Fc controls. (e) Western analysis of p-ERK and total ERK levels in NPC cultured on ephrinB2-Fc or Fc ligands for 24h. (f) Quantitative RT-PCR analysis of cyclinD1 and cyclinE mRNA levels in NPC stimulated with ephrinB2-Fc or Fc proteins (n=5 RNA extracts from independent experiments). Error bars denote s.d. Two-tailed paired Student’s t-test. (g) Quantification of NPC cell cycle profiles by FACS. Cells were seeded on Fc or ephrinB2-Fc for 24h and pulsed with BrdU for 1h (n=3 dishes from independent experiments). Error bars denote s.e.m. Each dot represents an independent experiment. One-way ANOVA with Bonferroni correction. (h) Quantification of FACS cell cycle profiles of NPC in monoculture (−) or coculture with wild type (+WT pEC) or Efnb2 knock-out (Efnb2−/− pEC) endothelial cells, measured by FACS (n=5 independent experiments). Error bars denote s.e.m. One-way ANOVA with Bonferroni correction. Source data in Supplementary table 1. Scale bars: c=30μm; d=50μm. Uncropped images of blots are shown in Supplementary Fig. 7.