Fig. 4

Iron accumulates in mitochondria of SFXN2-KO cells. a Genotyping of SFXN2-KO and control cells. b Alignment of DNA sequences of control and SFXN2-KO cells. Green letters show DNA regions corresponding to exon 4 and exon 5 of SFXN2. Grey box shows the deleted region in SFXN2-KO cells. c The mRNA level of SFXN2 was significantly lower in SFXN2-KO cells than in control cells. Expression of SFXN1, SFXN3, SFXN4, and SFXN5 did not differ between SFXN2-KO and control cells. n = 3 each. ***p < 0.001. d The mitochondrial iron content was measured using ICP-MS. This content was significantly higher in SFXN2-KO cells than in control cells. n = 3 each. **p < 0.01. e The mitochondrial iron contents of control and SFXN2-KO cells were investigated using Mito-FerroGreen. Bar = 10 μm. f The fluorescence intensity of Mito-FerroGreen was significantly higher in SFXN2-KO cells than in control cells. n = 100 control cells and 200 SFXN2-KO cells. ****p < 0.0001. g, h Control and SFXN2-KO cells were transfected with Mito-DsRed (g) or SFXN2-mCherry (h), and iron was stained with Mito-FerroGreen. i Quantification of the fluorescence intensity of Mito-FerroGreen in mock transfected cells and in cells transfected with Mito-DsRed and SFXN2-mCherry. n = 10–12. ****p < 0.0001. Expression of SFXN2-mCherry, but not of Mito-DsRed, suppressed iron accumulation in SFXN2-KO cells