FIGURE 6.

Wnt/NR2f2 signaling is responsible for transcriptional upregulation of GPX4 in BM. (A) Representative Western blot image showing the nuclear NR2F2 expression in PC9, PC9‐BrM3 cells, and PC9‐BrM3 cells treated with Wnt inhibitor IWR‐1‐endo for 48 hours. Histone H3 acted as the internal reference protein for nuclear protein. (B) Representative immunofluorescence staining images show the expression and location of NR2F2 in PC9, PC9‐BrM3 cells, and PC9‐BrM3 cells treated with Wnt inhibitor IWR‐1‐endo for 48 hours. Cell nuclei were stained with DAPI (blue) in PC9 cells with GPX4 overexpression (scale bar, 25 μm). (C) Results of Western blot showing the expressions of NR2F2 and GPX4 in PC9 cells treated with Wnt agonist 1 (1, 5, 10 μM) for 48 hours. Representative images of each group from three independent experiments are presented. Results of qPCR (D) and Western blot (E) showing the mRNA and protein expressions of GPX4 in PC9‐BrM3 cells after treatment with the Wnt inhibitor IWR‐1‐endo (10 μM) with or without transfection with NR2F2 plasmid, or transfected with NR2F2 siRNA or NR2F2 plasmid alone for 48 hours. Representative images of each group are presented and the bar graphs are summarized results from three independent experiments (n = 3, **P < 0.01). (F) Schematic illustration of a series of incremental deletion structures of the luciferase reporter gene (left) and the results of luciferase assays (right) where 293T cells were transiently transfected with the indicated pGL4 basic‐based reporter constructs for 24 hours and then the luciferase activity was measured (n = 3, ****P < 0.0001). (G) ChIP assay of NR2F2 binding to the GPX4 promoter. Lane 1, DNA marker; lane 2, input DNA; lane 3, DNA from PC9‐BrM3 cells immunoprecipitated with normal IgG; lane 4, DNA from PC9‐BrM3 cells immunoprecipitated with anti‐NR2F2 antibody. Representative images of each group from three independent experiments are presented. (H) Schematic illustration of the specific NR2F2 binding sites of GPX4 promotor (WT promoter) and the mutant sites (MUT promoter) for dual‐luciferase assays (upper). Results of luciferase assays (lower): 293T cells were transiently transfected with the indicated pGL4 basic‐based reporter constructs for 24 hours and then the luciferase activity was measured (n = 3, ****P < 0.0001). (I) Results of EMSA and supershift assay of NR2F2 binding to GPX4 promoter