Fig 2: Association of S100P with cell membranes. (A) Total cell extracts and membrane fractions prepared as described in Materials and Methods from Rama 37 cells expressing S100P proteins and were subjected to Western blotting to detect S100P protein, the cytoplasmic marker, tubulin, and the plasma membrane marker, caveolin 1. (B) Living cells not expressing S100P (Rama 37 + vector control; panels a–c) or expressing wild-type S100P (Rama 37 + S100P; panels d–f), K95A mutant S100P (Rama 37 + S100P K95A; panels g–i), or S100P protein with the C-terminal amino acid deleted (Rama 37 + S100P ?K95; panels j–l) were incubated with S100P antibody in the culture medium, and bound antibodies were detected with Cy-3-labelled secondary antibody, as described in Materials and Methods and observed with a Zeiss LSM510 confocal laser scanning microscope. Three separate typical fields are shown for each cell line. (C) Antibodies directed at S100P (panels a,f) or cytoplasmic protein, eEF1A (panels c,h), were added to the culture medium of living S100P-negative Rama 37 cells (Rama 37 + vector control; panels a,c) or S100P-positive Rama 37 cells (Rama 37 + S100P; panels f,h). Parallel cultures of S100P-negative cells (Rama 37 + vector control; panels b,d,e) and S100P-positive cells (Rama 37 + S100P; panels g,i,j) were permeabilised before being treated with the antibodies directed at S100P (permeabilised; panels b,g) or eEF1A (permeabilised; panels d,e,i,j). Following fixation, bound antibodies were detected with fluorescein isothiocyanate-conjugated secondary antibodies (Materials and Methods) and observed with a Zeiss LSM510 confocal laser scanning microscope. Panels (e,j) show the same fields as panels (d,i), but the intensity of the fluorescence signal has been reduced to show individual cells. Bars, (B,C) = 50 µm.

Fig 3: Effect of extracellular addition of S100P on migration of S100P non-expressing cells in Transwell and scratch-wound migration assays. (a) Transwell migration assays were carried out as described in Materials and Methods. The number of S100P-negative Rama 37 empty-vector-transfected control cells passing through to the underside of the membrane in 16 h in the presence of 10 mM 6-ACA (Rama 37 + vector + 10 mM 6-ACA), R&D Systems polyclonal goat S100P-specific antibody, Cat. No AF2957 at a concentration of 0.2 ng/µL in the medium (Rama 37 + vector + S100P antibody), 50 µg/mL aprotinin (Rama 37 + vector + 50 µg/mL aprotinin), or 20 µg/mL a-2-antiplasmin (Rama 37 + vector + 20 µg/mL antiplasmin), either in the absence (No S100P) or in the presence (+ S100P) of 10 µg/mL recombinant S100P (rS100P) were counted and are plotted as a percentage of the mean of untreated Rama 37 + vector control cells (Rama 37 + vector control). Each box and whisker plot shows the dispersion of data from 60 separate data points from 4 independent experiments (control, 6-ACA, S100P antibody) or 10 to 30 data points from 2 independent experiments (aprotinin and antiplasmin) carried out at different times. (b) Rama 37 cells transfected with empty expression vector were subjected to scratch-wound assays in the absence of or in the presence of various concentrations of rS100P added to the extracellular medium and the time-to-scratch-closure recorded for 9 separate wells in two independent experiments carried out at different times. For both (a,b), p-values are indicated for comparison between the box and whisker plots beneath the ends of the horizontal lines as n.s. not significant (p > 0.05) or significant ** (p < 0.0001). In (a), for clarity, some p-value comparisons are shown without lines and in red colour, as follows: n.s., no-S100P controls for 10 mM 6-ACA, 50 µg/mL aprotinin, and 20 µg/mL a-2-antiplasmin are not significantly different from the no-S100P, no-addition control (p = 0.055, 0.946, 0.932, respectively, Dunnett post-hoc test), or * no S100P for 0.2 ng/µL S100P antibody, significantly faster than the no-S100P, no-addition control (p = 0.0002). In all boxes for (a,b), the black diamond and line show the median value; the cross shows the mean value. The white circles outside the whiskers denote outliers of >1.5 times the interquartile range.

Fig 4: Immunofluorescence localisation of NMMIIA and S100P in cloned transfected cells. Rama 37 cells transfected with empty expression vector (Rama 37 + vector control; (a–c)) or Rama 37 cells overexpressing wild-type S100P (Rama 37 + S100P; (d–f)), K95A mutant S100P (Rama 37 + S100P K95A; (g–i)), or ?K95 mutant S100P (Rama 37 + S100P ?K95; (j–l)) were grown on fibronectin-coated coverslips for 48 h prior to fixation, permeabilisation, and staining using secondary antibody coupled to fluorescein isothiocyanate for NMMIIA (a,d,g,j) or Cy-3 for S100P (b,e,h,k). Cells were then mounted and viewed using a Zeiss LSM510 confocal laser scanning microscope. Merged images (c,f,i,l) are shown with overlaps in yellow. Arrows in (a,c,g,j) show filamental structures along the edge of the cell. The arrow in (d) shows foci of NMMIIA staining near the leading edge of the cell, which did not co-localise with S100P (f). The arrows in (e,f) show a large focus of S100P co-staining with NMMIIA in the perinuclear region. Bars, 10 µm in all panels.

Fig 5: 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)