Fig. 2

Sema5A affects in vitro melanoma cell invasion, migration and vasculogenic mimicry. a Western blotting analysis of Sema5A expression in M14 cells after 48 h transfection with pooled oligonucleotide mix against Sema5A (si-Sema5A) or scramble target (si-Ctrl) sequences. β-actin was evaluated as control of equivalent transfer and loading. Reported images are representative of three independent experiments with similar results. b In vitro cell migration and c invasion assay performed in M14 cells transfected with si-Ctrl or si-Sema5A. Values are presented as percentage of migrated/invaded cells in si-Sema5A versus control cells. d In vitro cell migration and (e) invasion assay performed in M14 cells transfected with empty vector (Ctrl) or vector expressing the Sema5A protein (ECD-Sema5A). Values are expressed as a percentage of migrated/invaded cells in ECD-Sema5A versus control cells. b-e Data were expressed as average ± standard deviation. *p-values < 0.05. f Western blotting analysis of Sema5A, total and phosphorylated AKT, total and phosphorylated p44/42 protein expression in M14 cells transfected with Ctrl and ECD-Sema5A vectors. Representative images of three independent experiments are reported. β-actin or HSP70/72 expression was evaluated as control of equivalent transfer and loading. g In vitro cell migration performed in M14 cells transfected with Ctrl or ECD-Sema5A, untreated or treated with 10 nM Trametinib. Values are expressed as a percentage of migrated cells in ECD-Sema5A versus control cells. Data are expressed as average ± standard deviation. *p-values < 0.05. h Representative images and i quantification of capillary-like structure formation in M14 melanoma clones overexpressing Sema5A protein (FL-Sema5A/1 and FL-Sema5A/6) and corresponding control clone (empty). Data were expressed as average ± standard deviation. ***p-values < 0.001. Scale bar, 300 μm