Reagents and antibodies
3‑[4,5‑Dimethylthiazol‑2‑yl]‑2,5‑diphenyl tetrazolium bromide (MTT) was purchased from Amresco, Inc. (VWR International LLC, Seongnam, Republic of Korea). Deferoxamine mesylate, 5-fluorouracil (5-FU), poly-2-hydroxyethyl methacylate, and sulfasalazine were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), and RSL3 was purchased from APExBIO Technology (Houston, TX, USA). Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from Gibco (ThermoFisher Scientific, Seoul, South Korea), and B27 supplement was purchased from Invitrogen (Thermo Fisher Scientific). The ALDH activity assay kit (colorimetric) was purchased from Abcam (Cambridge, MA, USA).
The antibodies specific for β-actin (C-4) (diluted 1:2,000), c‑Myc (9E10) (diluted 1:1,000), E-cadherin (H-108) (diluted 1:1,000), EpCAM (c-10) (diluted 1:1,000), ferritin heavy chain (B-12) (FTH1; diluted 1:2,000), Oct3/4 (C-10) (diluted 1:1,000), SNAIL (G-7) (diluted 1:1,000), Sox2 (E-4) (diluted 1:1,000), transferrin receptor (H68.4) (CD 71, TfRC; diluted 1:2,000), and vimentin (V9) (diluted 1:1,000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); CD24 [M1/69] (diluted 1:1,000), CD44 (diluted 1:1,000), CD105 (diluted 1:1,000), EGFR [EP38Y] (diluted 1:1,000), glutathione peroxidase 4 (GPX4 [EPNCIR144]; diluted 1:1,000), Lgr5 [EPR3065Y] (diluted 1:500), and SLC40A1 (ferroportin, FPN; diluted 1:1000) were purchased from Abcam; and α-smooth muscle actin (diluted 1:2,000; Sigma-Aldrich), CD10 antibody (SP67) (ready for use; Ventana; Tucson, AZ, USA), CD44-variant 9 (CD44v9/1459) (diluted 1:1,000; Novus Biologicals, CO, USA), fibronectin (diluted 1:2,000; Cedarlane, Canada), PAX8 (MRQ-50) (ready for use; Roche Diagnostics, Seoul, South Korea), and SLC7A11 (cysteine/glutamate transporter (xCT); diluted 1:2,000; Alomone Labs, Jerusalem, Israel) were purchased as indicated.
Human kidney proximal tubule cells (HK-2) and RCC cells (Caki-1, Caki-2, SNU-333, SNU-349, and SNU-1272) were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea), and cultured according to the supplier’s protocol. The HK-2, SNU-333, SNU-349, and SNU-1272 cells were cultured in RPMI-1640 medium, and Caki-1 and Caki-2 cells were cultured in DMEM/high glucose. All media were from Welgene (Gyeongsan, Republic of Korea) and contained 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were grown in monolayers at 37 ℃ in a 5% CO2 incubator otherwise described.
The effect of drugs on cell viability was evaluated by MTT reduction to its formazan product. The cells were seeded in triplicate wells of 96-well plates (2 x 103 cells/well), and treated with 5-fluorouracil, RSL3, sulfasalazine, and deferoxamine at various concentrations. The cells were incubated for three days and then 10 µl of the MTT reagent (5 mg/ml in PBS) was added to each well, dissolved in DMSO for 15 min, and the MTT reduction was measured 3 h later spectrophotometrically at 595 using the absorbance at 620 nm as background by a VERSAmax microplate reader (Molecular Devices Korea LLC). The absorbance values obtained from the wells of the untreated cells represented 100 % cell viability and were used for comparison to the treated cells.
The sphere-forming assays were conducted as previously reported [21,22] with a slight modification. In brief, single-cell suspensions were plated in 96-well plates covered with poly-2-hydroxyethyl methacylate to prevent cell attachment, at a density of 5 x 102 cells in serum-free DMEM/F12 medium supplemented with 1% B27 supplement, 20 ng/ml EGF, and 20 ng/ml bFGF. After 15 days in culture, the number and the size of the spheres per well were acquired.
ALDH1 activity assay
The aldefluor assay was performed according to the manufacturer’s instructions (Abcam) . Briefly, we labeled one TEST and one CONTROL tube for each sample. We lysed the cells and add activated ALDEFLUOR reagent to the lysed cell suspensions and 5 μl of DEAB to the CONTROL tube and then transferred to the TEST tube, mixed and immediately transferred 0.5 ml of the mixture to the CONTROL tube. The test samples were incubated at 37 °C, centrifuged, and resuspended in assay buffer. Finally, ALDH activity was measured spectrophotometrically at 450 nm using a VERSAmax microplate reader (Molecular Devices Korea, LLC). The absorbance values obtained from the HK-2 cells represent 100% activity and were used for comparison to the RCC cells.
To obtain the intracellular proteins, cultured cells were harvested in M‑PER mammalian protein extraction reagent (Thermo Fisher Scientific) including 1% protease inhibitor cocktail set III (EMD Millipore), 0.5% phosphatase inhibitor cocktail 2, and 0.5% phosphatase inhibitor cocktail 3 (both from Sigma‑Aldrich). The protein concentrations were assessed using BCA protein assay (ThermoFisher Scientific) according to the manufacturer's instructions.
Electrophoresis of the protein in cell lysates was performed with the TGX Stain‑Free FastCast™ Acrylamide Starter Kit (Bio‑Rad Laboratories, Inc., Seoul, South Korea) using a Tris/glycine buffer system (Bio‑Rad Laboratories) and transferred onto PVDF membranes as previously described . The membranes were blocked with 5% skim milk for 1 h and then incubated with primary antibodies overnight at 4 °C. After washing, peroxidase anti‑mouse or anti‑rabbit IgG antibodies (Vector Laboratories, Inc., Seoul, South Korea) were applied for 1 h at room temperature. Next, Western Lightning Chemiluminescence Reagent (PerkinElmer, Inc., Daejeon, South Korea) was used to detect the proteins. Anti‑GAPDH antibody was used as a loading control on the stripped membranes. The bands were quantified using AzureSpot analysis software (version 14.2; Azure™ c300; Azure Biosystems, Inc. Dublin, CA, USA).
Orthotopic RCC model establishment
All animal experiments were conducted in accordance with and approved by the Jeju National University Institutional Animal Care and Use Committee (2019-0015).
Athymic Balb/c nude mice (7-week-old males) were purchased from OrientBio (Seongnam, Republic of Korea). To induce an orthotopic RCC model, the mice were divided into five groups (n = 5 mice/group) according to the different RCC cell lines (Caki-1, Caki-2, SNU-333, SNU-349, and SNU-1272), and anesthetized using an intraperitoneal injection of sodium pentobarbital (EntobarTM, Hanlim Pharm. Co. Ltd., Seoul, South Korea). Under aseptic conditions, a small longitudinal incision was made in the left lower back, and 2 x 105 cells in 20 µl of Matrigel (BD Biosciences, Becton Dickinson, Seoul, South Korea) were injected with a 27-gauge needle into the renal capsule. Thirty days following the injection, the mice were euthanized, the tumor masses explanted and fixed for 24 h in 4% paraformaldehyde, and the fixed tissues were dehydrated and embedded in paraffin wax. Sections (4 µm-thick) were cut with a microtome and prepared for hematoxylin/eosin (H/E) staining and immunohistochemistry.
Commercially available patient kidney tissue arrays (CL2) from SuperBioChips Laboratories (Seoul, Republic of Korea) were used for immunohistochemistry (IHC). GPX4 IHC was performed according to the supplier’s protocol. Each array contains 50 tumor sections and nine matched control sections. As a primary antibody, we used a polyclonal rabbit anti-human GPX4 antibody (1:2000, Abcam). GPX4 was visualized by staining with a biotinylated anti-rabbit secondary antibody (1:200, Vector Laboratories), developed with diaminobenzidine, and counterstained with hematoxylin. Blind-scoring for the GPX4 immunostaining was performed by two independent observers who first assessed the overall staining intensity on a single array. Next, we used a 4-step scoring scale to score the individual sections: 0 for absent or very low, 1 for mild staining, 2 for moderate staining, and 3 for strong staining.
For the primary antibodies, CD 10 and paired box G8 (PAX8), used for diagnosing RCC , we used the Benchmark XT staining system (Roche Diagnostics) on the orthotopic RCC masses, developed with DAB and counterstained with hematoxylin.
All data were compiled from a minimum of three replicate experiments. The data are expressed as mean values ± SD. Statistically significant differences (p < 0.05) were obtained using the Student t-test or one-way analysis of variance (ANOVA) with a Bonferroni post-hoc test (MS excel 2010).