Fig. 3.

Upregulated HEPH with high ferroxidase activity accumulates on the cellular membrane and leads to decreased LIP. a Immunofluorescence staining analysis of HEPH proteins in breast cancer cells transfected with G9a shRNA or treated with 5 µM UNC0638 for 24 h. Shown are representative sections. Scale bars, 10 μm. b Western blotting tested HEPH level in separated cell components of membrane and cytoplasm. c HEPH activity in G9a knockdown cells was measured by the oxidation of pPD and ferrozine assays. The homologue ceruloplasmin served as a positive control. d The cellular labile iron pool in G9a knockdown or inhibited cells was measured using the calcein-AM assay. The arrows indicate when the iron chelator was added. e The cellular labile iron pool in G9a-overexpressed cells was measured. f Western blotting tested HEPH overexpression in MCF-7 and MDA-MB-231 cells and the cellular labile iron pool in these cells were measured. All the results are presented as means ± SD from three independent experiments. Two-tailed unpaired Student’s T-test was performed. *P < 0.05, **P < 0.01 and ***P < 0.001