Fig. 4

Wnt inhibitory activity is evolutionarily conserved between zebrafish Gpx4b and human GPX4. (A) The phenotypes of embryos injected with 600 pg gfp or GPX4 mRNA were scored and presented following the criteria described in Fig. 3A. Results are from three independent experiments and the total embryo numbers are given at the top. (B) Human GPX4 alters the expression of chd and gsc in zebrafish embryos at 4.3 hpf. Embryos injected with 600 pg gfp or GPX4 mRNA were analyzed by whole-mount in situ hybridization. The frequency of embryos with the indicated phenotypes is shown in the bottom right corner of each group. (C) Effectiveness of GPX4 shRNA at the protein level in HEK293T cells. shCtrl, control shRNA; β-tubulin, internal control. (D) Knockdown of GPX4 enhances endogenous Wnt signaling in vitro. The stable control or GPX4 knockdown lines were transfected with TOPFlash reporter DNA. (E) Stable knockdown of GPX4 in HEK293T cells increased the expression of the indicated Wnt direct target genes. Expression levels were analyzed by qRT-PCR. Values are means±s.e.m. (n=3). *P<0.05; **P<0.01; ***P<0.001. Unpaired t-test, two-tailed. Scale bar: 200 µm.