Fig. 3

S100P downregulation elicits different cellular behaviours in an E-cadherin dependent manner. a Cell-cell adhesion ability, evaluated by the slow aggregation assay, is compromised in MKN74 cells following transfection with siS100P. The graph shows quantification of aggregate area at 24 and 92 h. b Matrigel invasion assays were performed for MKN74, NCI-N87, KATOIII and MKN45 cells following transient transfection with siS100P (or with non-targeting siRNA). The graphs depict the relative number of invasive cells ±SD. Upon S100P downregulation, the invasive capacity increases in MKN74 and NCI-N87 cells but decreases in KATOIII and MKN45 cells. Apoptosis was evaluated upon depletion of S100P in MKN74 (c) and KATOIII (e) 48 h post transfection. Briefly, cells were stained with FITC Annexin V and propidium iodide (PI) and relative apoptosis levels were measured by flow cytometry, indicating a significant increase in the levels of apoptotic cells in KATOIII. d The levels of phosphorylated AKT were analyzed by Western blot, indicating increased activation in MKN74 cells. f Phosphorylated AKT and ERK expressions were evaluated by Western blot in KATOIII cells. Silencing of S100P did not alter AKT expression but led to a decrease in ERK activation. g Self-renewal potential, determined by the sphere-formation assay, increases in MKN74 cells and decreases in KATO III upon S100P downregulation. Sphere-forming efficiency is calculated based on the number of spheres divided by the number of cells plated. Data correspond to mean value ±SD and images are representative of at least three independent experiments. Statistical significance was evaluated with the Student’s t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001)