Figure 3.

Cellular levels of AGR2 correlate with pancreatic cancer cell proliferation, invasion, and survival in vitro. A, Western blot showing the effects of AGR2 silencing in cell lysates of MPanc-96 cells stably transfected with shControl or shAGR2-1, or shAGR2-2 vector. AGR2 was silenced in MPanc-96 cells. The micrograph is representative of three independent experiments. A full-length gel is presented in Supplementary Fig. S5. Effects of stable silencing of AGR2 on MPanc-96 cells were determined. B, MPanc-96 cells stably transfected with shControl or shAGR2-1 or shAGR2-2 (1,000 cells) were plated on 96-well plates with 0.5% serum containing DMEM, and cell numbers were estimated at 48 h by MTS assay. Stably silenced AGR2 cells showed a significant reduction in cell proliferation. Points, mean for three experiments; bars, SE (*, P < 0.05 versus control). C, effects of stable silencing of AGR2 on the invasiveness of MPanc-96 cells were analyzed. MPanc-96 cells stably transfected with shControl or shAGR2-1 or shAGR2-2 vectors were added into Biocoat Matrigel invasion upper chamber, and cells invading the lower chamber were estimated after 24 h using MTS reagent. Cellular invasiveness was significantly reduced on stably silenced AGR2 cells. Columns, means for three experiments; bars, SE (*, P < 0.05 versus control). Representative micrographs of invading cells in microscope fields for each membrane were pictured at 20× magnification. D, the effects of stable silencing of AGR2 on cell survival after gemcitabine treatment were examined. MPanc-96 cells stably transfected with shControl or shAGR2-1 or shAGR2-2 vectors were plated on 100-mm dish and gemcitabine (1 µmol/L) was added, and after 72 h, apoptotic cells were analyzed by FACS. Stable silencing of AGR2 showed significant increase in the gemcitabine sensitivity, and therefore, increase in apoptosis was observed. Columns, mean for three experiments; bars, SE (*, P < 0.05 versus control).