Figure 2.

Transient silencing of AGR2 reduces pancreatic cancer cell proliferation, invasion, and apoptosis. A, to access the effectiveness of siAGR2, AGR2 levels were determined in pancreatic cancer cells after being transfected with siControl or siAGR2 at a concentration of 10 nmol/L. Cell lysates were prepared after 72 h, and Western blotting was performed with an anti-AGR2 antibody and anti–β-actin as loading control. AGR2 was either partially or completely silenced in pancreatic cancer cell lines. The micrograph shown is representative of three independent experiments. A full-length gel is presented in Supplementary Fig. S4. B, to determine the effects of AGR2 on cell proliferation, pancreatic cancer cell lines transfected with siControl or siAGR2 were plated into 96-well plates, and cell numbers were estimated by MTS assay after 48 h. AGR2 silencing resulted in significant reduction in cell proliferation. C, for investigation of the effects of AGR2 on cell invasion, cells were transfected with siControl or siAGR2 and plated into Boyden chambers for invasion studies. After 24 h, the invaded cells in 10 random fields at 100× magnification were counted. AGR2 silenced cells showed a significant reduction in cell invasion. D, for apoptosis studies, cell lines were transfected with siControl or siAGR2 and were then treated with or without gemcitabine (1 µmol/L). After 72 h, the percentage of cells with a sub G₀/G₁ DNA content was identified by FACS analysis. AGR2 silencing resulted in increasing the sensitivity of the cells to gemcitabine and, therefore, resulted in increased apoptosis. Columns, mean for three experiments; bars, SE (*, P < 0.05 versus control).