Figure 4. NV669 affected the expression of cell adhesion molecules and induced cell detachment.

(A) Expression of PTP1B in BxPC3, MiaPaCa-2, HepG2 and Huh7 cancer cells lines. (B) Recombinant human PTP1B or (C) Tc-PTP were incubated in a microplate with 75 μM of phosphopeptide IR5 insulin receptor β-subunit domain and with increasing doses of NV669 (dark grey columns) or claramine (light grey columns), for 30 min at 30°C. Cells were also incubated with suramin (10 μM, white column) as positive control or spermine (150 μM, black column on the right) as negative control, for 30 min at 30°C. A colorimetric assay allows to determinate the released phosphate during enzymatic reactions. Values are means +/- SD of three triplicate experiments per conditions. (D) Cell morphology change after NV669 treatment was assessed using phase-contrast microscope (scale bar = 250 μm). (E) Expression of FAK, cadherin-1 (CDH-1), cadherin-2 (CDH-2), cadherin-3 (CDH-3) and β-catenin is determined by western blot after treatment with IC₅₀ concentration of NV669 for 24 h. β-actin was used as loading control. (F, G) The relative expression of these proteins was calculated by Image J software for BxPC3 (F) and Huh7 (G) cells. Values correspond to band intensity reported to actin band intensity and expressed as % of control values (without NV669 treatment). (H, I) Effects of NV669 at IC₅₀, 24h on cell detachment. Values are means +/- SD of three independent experiments. ^****, p<0.0001; ^***, p<0.001; ^**, p<0.01 and ^*, p<0.5; n.s, not significant.