Fig 2: N-cadherin and α9-integrin immunocytochemistry. Immunocytochemistry with anti-Hes1 and N-cadherin (N-Cad) antibodies (A) or Hes1 and α9-integrin (ITGA9) antibodies (B) in cells transfected with Delta or control vector (CTRL). Staining was as follows: Hes1 (green), N-cadherin and α9-integrin (red) and nuclei (blue). (C) Relative fluorescence quantification for Hes1, N-cadherin and α9-integrin. Student’s t-test significance: *P<0.05.

Fig 3: Genetic modifications of the Notch pathway influenced N-cadherin and α9-integrin expression. Western blots showing the concomitant variations of Notch pathway activation (NICD and Hes1) and N-cadherin (N-Cad/FL and CTF2) and α9-integrin (ITGA9) expression in RMS cells transfected with Delta (A) or dnMAML1 (B). Below each western blot composition, a plot depicting densitometric analysis of each band of the western blot. The variations associated with genetic modification of the Notch pathway (Delta and MAML1 transfections) observed in protein are also seen in the mRNAs of N-cadherin, α9-integrin and Hes1 determined by quantitative PCR (C). All RNA measurements were taken in triplicate. Student’s t-test significance: *P<0.05, **P<0.005.

Fig 4: Pharmacological inhibition of Notch signalling significantly reduced N-cadherin and α9-integrin expression. (A) Complementary DNA (cDNA) expression microarray showing the relative levels of α9-integrin (ITGA9), N-cadherin (N-Cad) and Hes1 expression in GSI-treated samples (GSI) compared with the control (DMSO). (B) Panel of western blots showing the effects of GSI on full-length N-cadherin (N-Cad/FL) and CTF2 fragment, α9-integrin (ITGA9), NICD and Hes1 levels compared with untreated samples (DMSO). Alpha-tubulin was used as a loading control. Below the western blot composition, a plot depicting densitometric analysis of each band of the western blot. (C) Relative mRNA levels evaluated by quantitative PCR in the presence (GSI) or absence (DMSO) of γ-secretase inhibitor. Neuronal cadherin, α9-integrin and Hes1 mRNA levels were evaluated in triplicate. Student’s t-test significance: *P<0.05, **P<0.005.

Fig 5: Anti-N-cadherin- and anti-α9-integrin-blocking antibodies reduced invasive properties induced by Notch activation. Three RMS cell lines were transfected with the constructions indicated below the bottom of each bar (pCDNA, Delta) in the presence (+) or absence (−) of either N-cadherin-blocking antibody (N-Cad Ab) or α9-integrin-blocking antibody (α9-ITG Ab) as indicated in the figure. (A) Adhesion assay on fibronectin-coated plates. The percentage of cells attached was quantified per cell line and condition. (B) Wound-healing assay. All bars indicate the healing velocity of each cell line, transfection and treatment (C) Matrigel/Transwell assay. Invasiveness is represented as a percentage of cells in the lower compartment, compared with the control. The three assays were performed in three independent wells per cell line and condition. Student’s t-test significance: *P<0.05.