Fig. 7

GPX4/Gpx4b binds to and prevents Tcf/Lef binding to target promoters. (A) Distribution of endogenous GPX4 protein in HeLa cells, visualized by immunofluorescence (green) with an anti-GPX4 antibody. Nuclei (blue) were stained with DAPI. Scale bar: 75 μm. (B) Endogenous GPX4 interacts with Tcf/Lef members, as indicated by co-immunoprecipitaton. Left panel: GPX4 interacts with TCF3/4 in HEK293T cells. Right panel: four Myc-tagged Tcf/Lef members interact with endogenous GPX4 in HEK293T cells. The indicated proteins were immunoprecipitated or detected using appropriate antibodies. Vector, empty expressing plasmid with myc tag; WCL, whole-cell lysate; IP, immunoprecipitate; input, positive control; IgG, negative control; arrow, IgG heavy chain. (C) Exogenous Gpx4b interacts with Tcf3. Proteins were extracted from lysates of cells that co-expressed tagged Tcf3 and Gpx4b, immunoprecipitated and subjected to western blot analysis using the indicated antibodies. (D) Gpx4b interacts with each indicated form of Tcf3 in living cells, as indicated by BiFC assay. Flag-tagged Gpx4b-VC expression vector was co-transfected with the indicated forms of Tcf3-VN. Expression of Gpx4b detected by anti-Flag antibody immunostaining (red). NC, negative control. Scale bar: 50 μm. (E,F) Zebrafish Gpx4b and human GPX4 inhibit VP16-Tcf3(295-441) action in vivo. One-cell stage wild-type embryos were injected with indicated mRNAs [680 pg gfp mRNA alone or 80 pg vp16-tcf3(295-441) plus 600 pg gfp, gpx4b or GPX4 mRNA]. Representative images of each group of injected embryos at 12.5 hpf are shown in E. Quantitative results of the indicated phenotypes are shown in F. The frequency of embryos with the indicated phenotypes is shown in E. Scale bar: 200 μm. (G) Gpx4b inhibits VP16-Tcf3(295-441)-induced activity in vivo. Embryos were injected with TOPFlash reporter DNA with the indicated mRNAs [680 pg gfp mRNA alone or 80 pg vp16-tcf3(295-441) plus 600 pg gfp or gpx4b mRNA]. (H) Endogenous GPX4 is associated with the promoters of the indicated direct Wnt target genes in HEK293T cells, as indicated by ChIP assay. α-Satellite and GAPDH promoters were used as negative controls. (I) Knockdown of GPX4 enhances the binding of TCF3/4, TCF4 alone and LEF1 to the AXIN2 and DKK1 promoters in HEK293T cells. Values are means±s.e.m. (n=3). *P<0.05; **P<0.01; ***P<0.001. Unpaired t-test, two-tailed.