Fig. 3: O-GlcNAcylation regulates cellular iron flux.

a U2OS cells were treated with DMSO (Control) or OSMI-1 for 24 h, and the relative cellular labile iron levels were assessed using Calcein-AM. b U2OS cells were treated as indicated for 24 h and cell lysates were subjected to immunoblotting with antibodies against IRP2 and β-actin. c, d U2OS cells were treated with DMSO (Control) or OSMI-1 for 12 h, and then treated with RSL3 for different time as indicated. The levels of cellular ferrous iron were assessed by flow cytometry using FerroOrange (c). Then the mean FerroOrange fluorescence intensity of each cell was quantified (d). e U2OS cells were treated with OSMI-1 or TMG for 24 h, and the cellular ferrous iron levels were assessed by 3D-structured illumination microscopy using FerroOrange. f U2OS cells were treated with OSMI-1 or TMG for 24 h, and the mitochondrial ferrous iron levels were assessed by confocal microscopy using Mito-FerroGreen. g U2OS cells were treated with OSMI-1 or TMG for 24 h, and the mitochondrial ferrous iron levels were assessed by flow cytometry using Mito-FerroGreen. Then the mean Mito-FerroGreen fluorescence intensity of each cell was quantified. h U2OS cells were treated with DMSO (Control) or OSMI-1 for 12 h, and then induced with RSL3 for different time as indicated. The mitochondrial ferrous iron levels were assessed by flow cytometry using Mito-FerroGreen. i U2OS cells were transfected with control or NCOA4 siRNAs for 48 h, and treated with DMSO (Control) or OSMI-1 for 24 h. The mitochondrial ferrous iron levels were assessed by confocal microscopy using Mito-FerroGreen. j U2OS cells were treated as described in i. The mitochondrial ferrous iron levels were assessed by flow cytometry using Mito-FerroGreen and then quantified. Scale bars, 10 μm. *P < 0.05, **P < 0.01, ****P < 0.0001. Error bars indicate SD.