Figure 3.

SEMA6D promotes tsA58-AVM cell migration. ( A , B ) tsA58-AVM cells were starved overnight and re-suspended in either conditioned medium (containing the ectodomain of SEMA6D) or control medium and subjected to the transwell migration test. The migrated nuclei were visualized with DAPI staining. For each filter, the number of migrated cells was determined as the sum of cells under three random fields (under a 10× objective). For each condition, data were averaged from six independent experiments performed in triplicate. The mean value of migrated cells treated with control medium is 221. ** P <  0.01, Student’s t -test. ( C , D ) tsA58-AVM cells were transfected with a scrambled siRNA control or two independent siRNAs against Sema6D . Two days after transfection, cells were treated with 0 ng/ml or 100 ng/ml BMP4 for 48-h followed by transwell migration analysis ( C ). The results from only one siRNA (si-S6D-1) treatment are shown in panel C . Panel D shows the quantification results averaged from six independent samples. The mean value of cells transfected with the scrambled control and without BMP4 treatment is 235. Two-way ANOVA reveals the effect of siRNAs and BMP4 (see Supplementary material online, Table S3 ). **** P <  0.0001, by post hoc Sidak’s multiple comparisons test. The bars in A and C represent 200 µm. Error bars in B and D indicate SEM.