Figure 4

MiR-362 activates the NF-κB signaling pathway. (A) NF-κB luciferase reporter activities were analyzed in cells in which miR-362 had been transfected or inhibited (miR-362-in). (B) Relative mRNA expression of NF-κB–regulated genes in cells. GAPDH was used as a loading control. (C) Western blotting of total and nuclear expression of p65. α-Tubulin and the nuclear protein p84 served as loading markers. (D) Immunofluorescence staining of subcellular localization of NF-κB p65 in cells. (E) Annexin V–FITC/PI staining of BGC-823 cells transfected with miR-362 or miR-362 plus IκBα dominant-negative mutant (IκBα-mut) and treated with 20 μM cisplatin for 24 h. (F) Flow cytometric analysis of BGC-823 cells transfected with miR-362 or miR-362 plus IκBα-mut. Bars denote the mean of three independent experiments. *P < 0.05. NC, Negative control.