Silencing of <i>PTGR2</i> promoted cell death and ROS production and induced 15-keto-PGE₂ in BxPC-3 cells.

<p><b>(A)</b> The percentage of dead cells in si-PTGR2 BxPC-3 cells as compared to si-Control cells was evaluated by Annexin V and 7-AAD staining. The flow cytometry plots show annexin V-FITC binding (FL1-H) and 7-AAD staining (FL3-H). The bar graph distinguish dead cells as apoptotic or necrotic. The results are the average of 4 independent experiments each done in triplicate. (<b>B)</b> Relative ROS production in si-PTGR2 (red profile) BxPC-3 cells as compared to si-Control (black profile) cells. ROS was detected using H₂DCF dye and flow cytometry. The results are the average of 3 independent experiments each done in triplicate. (<b>C)</b> Western blot analysis of the expression levels of COX1, COX2, 15-PGDH and PTGR2 in si-PTGR2 BxPC-3 cells. GAPDH served as a loading control. <b>(D)</b> Relative production of 13,14-dihydro-15-keto-PGE₂ in si-PTGR2 BxPC-3 cells as compared to si-Control cells. The concentration for si-Control cells was set as 1, and the relative levels of 13,14-dihydro-15-keto-PGE₂ in si-PTGR2 cells was presented as values relative to the control. The values were obtained from 2 independent experiments each done in triplicate. <b>(E</b> and <b>F)</b> Relative levels of intracellular <b>(E)</b> 15-keto-PGE₂ and <b>(F)</b> 13,14-dihydro-15-keto-PGE₂ isolated from si-PTGR2 BxPC-3 cells as compared to si-Control cells. The concentrations of 15-keto-PGE₂ and 13,14-dihydro-15-keto-PGE₂ extracted from si-Control cells were set as 1, and the relative levels of 15-keto-PGE₂ and 13,14-dihydro-15-keto-PGE₂ from si-PTGR2 cells were presented as values relative to the control. Prostaglandins were isolated and analyzed by LC-MS/MS. The results are the average of 3 independent experiments. Data are presented as the mean ± SE. * P < 0.05, ** P < 0.01, Student’s t-test.