Fig 2: TFR1 is the key gene leading to ferroptosis in CVB3-infected HeLa cells.A The mRNA level of TFR1 at different CVB3 infection times was detected via qPCR. The TFR1 mRNA increased obviously at 24 h of CVB3 infection (p < 0.001). B, C The protein level of TFR1 and relative gray values at different CVB3 infection times were measured via western-blotting. D TFR1 knockdown effects on three different sequences at the mRNA level via qPCR. E Determination of nuclear localization of TFR1 by immunofluorescence with CVB3 infection. The results showed that the TFR1 was translocated from the cell membrane to the nucleus with CVB3 infection. Scale bar = 10 μm. F, G The effect of ferroptosis drugs on TFR1 protein expression at 48 h post-CVB3 infection determined by western-blotting. H, I The image and gray values of TFR1 knockdown effects on three different sequences at the protein level via western-blotting. J Lipid ROS levels in siNC and siTFR1 groups were detected via immunofluorescence with BODIPY581/591C11 (scale bar = 100 μm). K The statistical results showed that the lipid ROS decreased significantly in siTFR1 group compared to siNC group (p < 0.001). L Subcellular fractionation analysis of TFR1 expressions in cell membrane, nuclear, cytoplasm using different cell component extraction kits and measured by western-blot. Na,K-ATPase, lamin b1 and β-actin were as internal references for cell membrane, nucleus, and cytoplasm in western-blot, respectively. M Cell viability in the siTFR1 group was obviously improved at 48 h post-CVB3 infection, measured by CCK-8 assays. N MDA levels were measured by MDA Assay Kit at a wavelength of 532 nm. It showed knockdown of TFR1 could reduce MDA levels compared to the wide-type group (p < 0.001). O The iron level in siTFR1 and NC groups were measured using an iron assay. It indicated that TFR1 knockdown could decrease cellular iron levels obviously. P, Q Effect of TFR1 knockdown on CVB3 fluorescence detected by IF. Cell nuclei were stained with DAPI (blue) (scale bar = 50 μm). R–T The mRNA levels of ferroptosis genes ACSL4, GPX4 and NCOA4 of TFR1 knockdown via qPCR. U, W The protein levels of major genes in the siTFR1 groups were detected via western-blotting. Corresponding of relative gray values of proteins were measured using ImageJ. V Relative gray values of subcellular fractionation analysis of western-blot strips in figure L measured by ImageJ. It suggested that membrane protein TFR1 was mainly located in membrane in mock group, however, it was partially transfered to nuclear induced by CVB3 infection, especially at 48 h postinfection.

Fig 3: TFRC transcriptional regulation.A Map of the promoter location in the TFRC gene. B Construction map of the TFRC promoter plasmid. C Construction map of the firefly luciferase reporter plasmid. D The activity of the TFRC promoter in the CVB3 group was significantly higher than that in the mock group based on luciferase reporter assay results. E mRNA level of Sp1 was consistent with that of TFRC at various CVB3 infection times by qPCR. F Effect of Sp1 knockdown and overexpression at the mRNA level determined by qPCR. G The influence of Sp1 upregulation and downregulation on TFRC mRNA expression via qPCR. H, I The effect of Sp1 upregulation and downregulation on TFRC protein expression. J Downregulation of Sp1 reduced the activity of the TFRC promoter by Luciferase Reporter Assay. K Upregulation of Sp1 enhanced the activity of TFRC promoter by Luciferase Reporter Assay. L Chromatin from HeLa cell lysis buffer was subjected to agarose electrophoresis before and after sonication in the ChIP experiment. M The three potential binding sites (BS) for Sp1 within the promoter region of TFRC predicted by JASPAR (http://jaspar.genereg.net/). N, O The binding sites for the transcription factor Sp1 in the TFRC promoter were verified via ChIP-PCR agarose gel electrophoresis (white arrows—indicate binding sites). The input group included total chromatin samples, and IgG served as the negative control. In the IP- Sp1group, the results showed that objective bends (150 bp) only emerged at BS1. All results are expressed as the mean ± SD. *p < 0.05 vs. the mock group, ** p < 0.01, ***p < 0.001. n = 3.

Fig 4: Butyrate-induced iron accumulation is dependent on ferritinophagy in PDLFs.A Labile iron pool was measured using PG SK method in control or butyrate (8 mM)-treated PDLFs. B Intracellular Fe2+ in control or butyrate (8 mM)-treated PDLFs were detected by FerroOrange. C PDLFs were pretreated with an iron chelator, deferoxamine (100 µM), for 2 h, and treated with butyrate (32 mM) for 36 h, then cell death was detected by LDH assay. D TfR, NCOA4, FPN, ferritin, P62, LC3B, and GAPDH expression in response to butyrate (8 mM) were assessed by Western blot. E Representative images of colocalization of ferritin (red) with LC3-GFP (green) in butyrate-treated PDLFs at 12 h. Scale bars = 20 µm.

Fig 5: Single injection of EPO increases TFR1 protein content in plasma exosomes. Immunoblotting for TFR2 and TFR1 in plasma exosomes isolated from control (C) and EPO (E) treated mice. EPO was administered at 200 U/mouse; mice were sacrificed 72 h after EPO injection. Arrow denotes the TFR1-specific band, arrowhead indicates the expected size of exosomal TFR2, which was not detected. The faint bands around 65 kDa in the TFR2 panel are probably nonspecific. GAPDH is used as loading control.