Figure 6

Changes in intracellular Ca ²⁺ levels in response to resveratrol. (A) Representative fluorescence images showing the level of intracellular Ca²⁺ in SHY_vector and SHY_TG2 cells. After 48 h resveratrol treatment in scratch assays, cells were labeled with Ca²⁺-indicator dye, Fluo4-AM, for 1 h, and examined under fluorescence microscope using FITC filter. Cells treated with resveratrol were also treated with cell permeable Ca²⁺ chelator, BAPTA-AM (10 μM) to chelate intracellular Ca²⁺ in cells. Scale bar = 100 μm. Fluorescence intensity in cells present near the scratch was measured using Image-Pro Plus software and plotted as fold change in IOD (B). (C) Representative immunofluorescence images for TG2 in scratch assays. SHY_TG2 cells were grown in chamber slides and scratch assays were performed in presence of Ca²⁺ chelator, BAPTA-AM (10 μM), alone or in combination with resveratrol (1 or 10 μM). After 48 h treatments, cells were labeled with anti-TG2 antibody (red) and images were taken randomly from the edge of the scratch using fluorescence microscope equipped with rhodamine filter. Enhanced red intensity in cells present near the scratch (white arrows) depicts increased immunoreactivity for TG2 that was attenuated in presence of Ca²⁺-chelator, BAPTA-AM. Scale bar = 100 μm. Fluorescence intensity in cells present near the scratch was measured using Image-Pro Plus software and plotted as fold change in IOD (D). (E) Representative Western blots for TG2 in presence of a Ca²⁺-chelator, BAPTA-AM, showing that resveratrol-induced slower mobility TG2 form is sensitive to Ca²⁺ levels. Native proteins isolated from resveratrol treated SHY_TG2 and Panc-28 cells in the presence or absence of BAPTA-AM (10 μM) from near the scratch were isolated, separated under non-denaturing conditions, and blotted with anti-TG2 antibody. The experiments were repeated three times.