Figure 5.

ER stress induces Ca²⁺ flux and increases tTG2 and PAD2 activity. A: To determine whether Thaps-induced ER stress leads to an increase in cytosolic Ca²⁺, βLox5 cells were labeled with the Ca²⁺ indicator Fluo-4 before incubation in medium alone (top row) or with DMSO (middle row) or Thaps (bottom row). B: A plot of the full time course of the Fluo-4 Ca²⁺ indicator clearly shows differences in Ca²⁺ flux elicited by Thaps treatment that are absent in the medium and the DMSO control (Ctrl.). C: The change in peak Fluo-4 intensity (from 0 to 203 s) was significantly higher for Thaps-treated cells vs. medium- or DMSO-treated cells. Data are the mean ± SEM of three independent experiments. D: Activity of tTG2 in Thaps- or DMSO-treated βLox5 cells was measured by the incorporation of biotinylated pentylamine into endogenous proteins. Cells incubated with 5 μmol/L Thaps exhibited 1.9-fold higher tTG2 activity than control-treated β-cells. Data are the mean ± SEM of five independent experiments. E: PAD2 expression in Thaps-treated βLox5 insulinoma cells was measured by Western blotting. Bands for one representative blot are shown (left). Densitometry data (right) are the mean ± SEM of seven independent experiments. F: Activity of PAD2 in Thaps- or control-treated βLox5 cells was measured by assessing the incorporation of citrulline residues in whole-cell lysates by Western blotting. Bands for one representative blot are shown (left). Densitometry data (right) are the mean ± SEM of five independent experiments. *P < 0.05.