Fig 2: Loss of a-syn alters levels of proteins involved in iron-homeostasis. (a) Representative Western blots of iron-related proteins (ferritin, a-syn, TfR1, and DMT1) in the indicated xenograft lysates. (b–d) Quantitative analysis of fold changes in ferritin, TfR1 and DMT1, derived from densitometric analysis of band intensities normalized to a-tubulin. P-values were determined using a one-way ANOVA with Dunnett posthoc test (n = 3). (e) Representative Western blots of ferroportin (FPN1) and a-syn in the indicated xenograft lysates. (f) Quantitative analysis of the fold change in FPN1, derived from densitometric analysis of band intensities normalized to a-tubulin. P-values were determined using a one-way ANOVA with Dunnett posthoc test (n = 3). Error bars in each plot are ± s.d. Uncropped sections of blots are shown in Supplementary Figure S5.

Fig 3: Hypoxia promotes iron mobilization from the intestinal mucosa. (A–C) Human subjects (n = 10) were exposed to hypoxic conditions in a hypobaric chamber simulating an altitude of 4000 meters above sea level for 3 hours. Distal colon biopsy specimens were taken the day before entering the hypobaric chamber (T0), immediately after hypoxia (T1), and 1 week after collection of the first biopsy (T2). Immunostaining for (A) ferritin, (B) DMT-1, and (C) FPN was performed and the areas of positive staining were quantified and normalized to nuclei staining. Results represent means + SEM. *P < .05; **P < .01. Scale bars: 25 µm. (D) Iron content in colon biopsy specimens also was assessed by atomic absorption spectrometry. (E–H) Transcript analysis for (E) ferritin, (F) DMT-1, (G) ferroportin, and (H) IL6 was performed. (I–L) Serum samples were collected at the time of screening (S), the day before entering the hypobaric chamber (baseline, T0), and 1 day (T1), 1 week (T2), and 1 month (T3) after baseline. Laboratory analysis of (I) serum iron, (J) Tf saturation, (K) Tf, and (L) ferritin was performed. Results represent means + SEM.

Fig 4: PEITC altered iron metabolism in human OS cells. (a) Total iron level in MNNG/HOS, U-2 OS, MG-63, and 143B cells after the indicated concentrations of PEITC treatment for 24 h by AAS. (b) The level of labile iron in MNNG/HOS, U-2 OS, MG-63, and 143B cells after the indicated concentrations of PEITC treatment for 24 h with Calcein-AM staining by flow cytometry analysis. (c) Protein expression levels of TfR1, DMT1, FTH1, FPN, and IRP2 in MNNG/HOS, U-2 OS, MG-63, and 143B cells treated with the indicated concentrations of PEITC for 20 h or 30 µM PEITC for 4 h, 12 h, 24 h, and 48 h. (d) mRNA levels of TFRC, SLC11A2, FTH1, SLC40A1, and IRP2 in MNNG/HOS, U-2 OS, MG-63, and 143B cells after PEITC treatment for 24 h. (e) Protein expression levels of NCOA4 in MNNG/HOS, U-2 OS, MG-63, and 143B cells treated with the indicated concentrations of PEITC for 20 h or 30 µM PEITC for 4 h, 12 h, 24 h, and 48 h. All data were presented as the means ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 versus control group.

Fig 5: Hypoxia induces the expression of iron transport proteins. (A–H) HT-29 cells and Caco-2 cells were subjected to normoxia (21% O2) or hypoxia (0.2% O2) for 24 hours in the presence or absence of 150 µmol/L DFO or 200 µmol/L FAC, and transcript analysis of (A and C) DMT-1, (B and D) FPN, (E and F) ferritin, and (F and H) vascular endothelial growth factor (VEGF) was performed. Results represent means + SEM of 3 independent experiments performed in triplicate. *P < .05; **P < .01; ***P < .001; ****P < .0001. (I and J) Total protein was isolated after 4 and 24 hours of hypoxia and Western blot analysis for HIF-1a, ferritin, and ß-actin was performed. Representative Western blots of 2 independent experiments. CTRL, control.