Ab30760

This study was approved by the Institutional Research Ethics Committee of HanDan Central Hospital. Written informed consent was obtained from the participants. Ten formalin-fixed, paraffin-embedded lung cancer tissues and normal lung tissues were used for IHC staining. Briefly, 4-μm sections of tissues were mounted on glass microscope slides, deparaffinized in xylene, and then rehydrated in sequentially increasing dilutions of alcohol. Antigen retrieval was performed at a high temperature using a water bath. The sections were cooled and rinsed, and endogenous peroxidases were quenched using 3% hydrogen peroxide. Then, the sections were washed three times with PBS, incubated with calf serum to block nonspecific antigens for 10 min, incubated with anti-hepcidin polyclonal primary antibody (1:200, ab30760, Abcam, Cambridge, MA, USA) overnight at 4°C, washed with PBS three times, and then incubated with secondary antibody for 30-40 min at room temperature (RT). Dried sections were observed with an optical microscope. The IHC staining results were analyzed and scored by two pathologists who were blinded to the sources of the clinical samples. A semiquantitative integration method was used to analyze the intensity of staining.

Protein samples were prepared from human serum (DS and age-matched controls, n = 20). Twenty μg protein samples were separated on 4–12% Nu-PAGE Bis-tris (Bis (2-hydroxyethyl)-amino-tris (hydroxymethyl)-methane) gradient gels and transferred to 0.2 μm pore size PVDF for Aβ42, hepcidin and S100β, and 0.45 μm pore size PVDF membranes for SOD1, FTL, FPN, TREM2 and albumin using the NuPAGE electrophoresis system (Invitrogen). Membranes were incubated with different antibodies, i.e., anti-β amyloid (Covance, SIG 39320), SOD1 (Abcam, Cambridge, UK, ab252426), S100β (Abcam, ab218514), anti-ferritin light chain (FTL, Abcam, ab69090), anti-FPN (Abcam, ab85370), anti-hepcidin 25 (Abcam, ab30760), anti-TREM2 (Abcam, ab86491) and anti-albumin (Abcam, ab10241) antibody, in blocking buffer for 24 h at 4 °C, and then washed three times with 0.1 M Tris saline buffer containing 1% Tween 20 (TBST), followed by incubation for 1 h at RT with HRP-conjugated secondary antibodies, anti-rabbit IgG (1:3000, DAKO) or anti-mouse IgG (1:3000; DAKO) antibodies. Binding was detected with ECL Plus chemiluminescence reagents.

Tissues were prepared as described previously (Lakhal-Littleton et al., 2015 (link)) and stained with rabbit polyclonal anti-mouse HAMP antibody (ab30760, Abcam, RRID:AB_2115844) at 1/40 dilution, or rabbit polyclonal anti-mouse FPN antibody (MTP11-A, Alpha Diagnostics, RRID:AB_1619475) at 1/200 dilution. Results of control experiments confirming the specificity of the HAMP antibody are shown in Figure 1—figure supplement 5.

We combined the gene expression data of control samples (17 samples) with those of CRC samples (533samples) in the GSE39582 group and performed the Wilcoxon rank-sum test to further compare the differential expression of the 10 prognostic ferroptosis-related genes between the normal and tumor colon tissues. Besides, a total of 75-paired normal/tumor CRC specimens were recruited from Ruijin Hospital (Shanghai, China) following the guidelines set by the Ethical Committee of Ruijin Hospital. The tumor and adjacent normal colon tissues were fixed by 10% formalin and embedded by paraffin. The optimum sections of tissue specimens were selected and deparaffinized and immunohistochemistry (IHC) was implemented as the following antibodies: HAMP (Abcam, ab30760), FDFT1 (Abcam, ab195046), GDF15 (Abcam, ab206414), TFAP2C (Abcam, ab218107).

Specific antibodies directed against p-Smad2 (3101, 1:2,000 dilution), p-Smad3 (9520, 1:2,000 dilution), Smad2/3 (3102, 1:2,000 dilution), Smad3 (9523, 1:2,000 dilution), p-Akt (9275, 1:2,000 dilution) and Akt (4685, 1:2,000 dilution) were purchased from Cell Signaling (Beverly, MA, USA). Anti-hepcidin and anti-collagen I (ab34710) antibodies were obtained from Abcam (Cambridge, MA, USA; ab30760 for human hepcidin or ab81010 for murine form). Anti-FPN (MTP11-A, 1:2,000 dilution) and anti-α-SMA (ab85370, 1:2,000 dilution) antibodies were supplied by Alpha Diagnostic International (San Antonio, TX, USA) and Abcam (Cambridge, MA, USA), respectively. Anti-β-actin antibody (A5441, 1:10,000 dilution) and other reagents were provided by Sigma-Aldrich (St. Louis, MO, USA). Synthetic human or murine hepcidin recombinant peptide (25 or 20 amino acids) and recombinant TGFβ1 were purchased from Peptides International (Louisville, KY, USA) and R&D Systems (Minneapolis, MN, USA), respectively.

Cells were washed with PBS and fixed for 30 min with 4% paraformaldehyde, permeabilized with 1% sodium dodecyl sulfate for 15 min and blocked with 1% bovine serum albumin for 1 h at RT. Primary antibody against hepcidin (Abcam ab30760) and the N-terminus of 24p3R^{31 (link)} were both diluted 1:100 in blocking solution and incubated overnight at 4 °C. The second antibody (Goat-anti rabbit, Invitrogen A-11008) was incubated for 1 h at RT diluted 1:600. DAPI (300 nM for 5 min) was used to counterstain nuclei. Hepcidin staining was visualized using a Zeiss ApoTome.2 microscope and imaged using Axiovision 4.8.

Fifteen µg of anti-hepcidin-25 (ab30760; Abcam, Cambridge, UK) antibodies were coupled with 50 µL of protein G + beads overnight at 4 °C. Twenty microlitre of serum provided by a NU patient was then incubated with beads for 2 h at 4 °C. After several washes with PBS containing 0.1 % of Tween-20, bound fractions were eluted with 100 mM acetic acid containing 30 % acetonitrile. Unbound (Flowthrough) and bound (Eluate) fractions were analyzed on IMAC-Cu²⁺ arrays by SELDI-TOF-MS. Non-specific IgG antibodies were used as negative controls.