Fig 2: Antioxidants Bypass the Requirement of FTH in the Establishment of Normoglycemia and Disease Tolerance to Sepsis(A–C) Relative weight (A), blood glucose (B), and survival (C) of Mx1CreFth?/? mice subjected to CLP and receiving NAC (n = 7; 15 mg/kg) or vehicle (n = 6). Data pooled from two independent experiments.(D) Colony forming units (CFU) for aerobic (Ae) and anaerobic (An) bacteria, 48 hr after CLP. PC, peritoneal cavity. Red bars represent mean values and doted circles represent individual mice. Pooled from two independent experiments.(E) Expression of G6PC1 mRNA quantified by qRT-PCR in HepG2 cells exposed (+) or not (-) to heme, TNF, and/or NAC. Data shown as mean ± SD from four independent experiments.(F and G) Blood glucose (F) and survival (G) of Mx1CreFth?/? mice subjected to CLP and receiving BHA (n = 11; 50 mg/kg) or vehicle (n = 9). Data shown as mean ± SD. Pooled from three independent experiments.(H–J) Relative weight (H), blood glucose (I), and survival (J) of AlbCreERT2G6pc1?/? mice subjected to CLP and receiving NAC (n = 5) or vehicle (n = 5). Mean ± SD from one experiment.NS, non-significant. *p < 0.05, **p < 0.01. Numbers in gray (A, B, F, I, and J) indicate live/total mice.

Fig 3: FTH Regulates Glucose Metabolism in Response to Polymicrobial Infection(A–C) Relative weight (A), temperature (B), and blood glucose levels (C) in Fthlox/lox (n = 13) and Mx1CreFth?/? (n = 15) mice subjected to CLP. Mean ± SEM from five independent experiments.(D) Blood glucose levels in Mx1CreFth?/? mice subjected to CLP and receiving glucose (n = 15) (gavage; 2 mg/kg body weight [BW]; two times daily for 4 days and one time daily for 3 days) or vehicle (n = 12). Mean ± SEM from three independent experiments.(E) Survival of the same mice as in (D).(F) CFU for aerobic (Ae) and anaerobic (An) bacteria, 48 hr after CLP. Red bars represent mean values and dotted circles individual mice.(G) Blood glucose levels in C57BL/6 mice receiving heme (n = 15) (intraperitoneally [i.p.] 30 mg/kg BW) or control protoporphyrin IX (PPIX) (n = 10). Mean ± SEM from three to five independent experiments.(H) Blood glucose levels in Fthlox/lox (n = 8) and Mx1CreFth?/? (n = 9) mice receiving heme (i.p. 25–30 mg/kg BW). Mean ± SD from two independent experiments.(I) Survival of Fthlox/lox (n = 8) and Mx1CreFth?/? (n = 9) mice receiving heme (i.p. 25–30 mg/kg BW).(J) Blood glucose levels in C57BL/6 Tlr4+/+ (n = 13) and Tlr4-/- (n = 9) mice receiving heme (i.p. 30 mg/kg BW). Mean ± SEM from three to four independent experiments.(K) Blood glucose levels in C57BL/6 Ifnr1+/+ (n = 7) and Ifnr1-/- (n = 7) mice receiving heme (i.p. 30 mg/kg BW).Mean ± SD from two independent experiments. PC, peritoneal cavity. Numbers in gray in (A)–(D) and (H) are live/total mice at each time point. *p < 0.05; **p < 0.01.See also Figure S4.

Fig 4: Radiation induced Lysosomal membrane permeabilization (LMP) in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were treated with 4 Gy, 8 Gy radiation respectively, the amount of LMP was detected at 4, 8 h with LysoTracker and analyzed by flow cytometry (A–C) or with AO staining (D). In the presence or absence of NH4Cl, the Ferritin (FTH) expression were detected after 8 Gy treatment at 24 h (E). Cells were pretreated with 3MA (2 mM) or MG132 (1 μM) for 1 h before 8 Gy radiation, the Ferritin (FTH) expression were detected at 24 h (F). Cells were pretreated with 3MA before 8 Gy radiation, the intracellular iron was evaluated with Prussian blue staining at 24 h by light microscopy (G). These results were representative of three independent experiments and expressed as means ± SD. P-values were calculated using repeated measure ANOVA with P < 0.05 being considered as statistical significance, *P < 0.05.

Fig 5: VAMP2 is involved in EA-regulated FPN translocation.(A) mRNA expression levels of Vamp (Vamp2, Vamp8 and Vamp8), SNAP23 and syntaxin 4 in primary HSCs from C57BL/6 mice and LX-2 cells. n = 4. (B) Protein expression of VAMP2, VAMP3, VAMP8, syntaxin 4, and SNAP23 in primary HSCs and LX-2 cells treated with TGF-β1 and/or EA. n = 5. (C) VAMP2 expression in primary HSCs and LX2 cells pretreated with MG-132 (10 μM) or CQ (1 μM) for 30 min followed by TGF-β1 and/or EA treatment. n = 5. (D) Following TGF-β1 treatment, VAMP2 expression was analyzed in primary HSCs and LX2 cells pretreated with CHX (100 μg/mL) for the indicated durations. n = 5. (E) Immunoblotting analysis of syntaxin 4 and SNAP23 in VAMP2 immunoprecipitates from primary HSCs and LX2 cells treated with TGF-β1 and/or EA. n = 4. (F) Primary HSCs and LX-2 cells were infected with different concentrations of AdshVAMP2, AdVAMP2 or control adenovirus (AdLacz) for 24 h. VAMP2 protein expression was examined. n = 5. (G) After HSCs were infected with AdshVAMP2 or AdVAMP2 followed by TGF-β1 treatment, the cells were incubated in EA for 24 h, and then the distribution of FPN and (H) FTH1 expression in those cells was examined by western blotting. n = 5. Data were normalized to GAPDH and are expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 between the indicated groups.