Fig. 3

EphrinA5 stimulation leads to reduced association of DNMT1 to Ncam1 and diminished motility of CB cells. a, b The motility of CB cells is significantly reduced upon stimulation with ephrinA5-Fc, which can be rescued by a preceding knockdown of Ncam1. a Temporal color-coded migratory distance over 20 h of imaging. The starting point of migration for each cell is shown in dark blue and the end point in white. b Quantitative analysis of average migratory speed (n = 557 for ctrl siR + ctrl-Fc, n = 455 for ctrl siR + efnA5-Fc, n = 481 for Ncam1 siR + ctrl-Fc, n = 495 for Ncam1 siR + efnA5-Fc, N = 4 biological replicates). c-f Native ChIP revealed decreased enrichment of DNMT1 in the Ncam1 promoter region close to putative Snhg15-binding sites. c Genomic map depicting the promoter region of the murine Ncam1 gene. Regions targeted by the primer pairs P1, P2 and P3 are shown in turquoise, the promoter in dark blue, candidate cis-regulatory elements in red, CpG site with a significantly reduced methylation level in black, and putative Snhg15-binding sites in pink. d-f ChIP-qPCR analysis using anti-DNMT1 and anti-H3K27me3 antibodies normalized against the input material and IgG (N = 4 biological replicates). Significances were determined with one-way ANOVA (b) and two-tailed Student’s t-test (d-f). Significance levels: p value < 0.05 *; p value < 0.01 **; p value < 0.001 ***. Scale bar: 100 μm. ctrl: control. efnA5: ephrinA5. siR: siRNA