Data processThe expression profiles associated with ESCC (GSE75241) were retrieved from the Gene Expression comprehensive database (GEO) (https://www.ncbi.nlm.nih.gov/geo/). GSE75241 contained RNA expression data annotated by GPL5175, which included 15 paired ESCC samples and matched nonmalignant mucosa.
Cell lines
The human esophageal squamous cell carcinoma (ESCC) cell lines KYSE450, KYSE150, KYSE30, KYSE510, KYSE410, KYSE140, and KYSE70 were obtained from the Chinese Academy of Sciences cell bank (Shanghai, China). The normal esophageal cell line SHEE and the embryonic kidney cell line HEK293T was obtained from Shantou University (Shantou, Guangdong, China). All cells were cultured at 37°C in a humidified atmosphere with 5% CO2. RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Biological Industries) and penicillin (100 U/ml) and streptomycin (100 ng/ml) (Meilunbio, Dalian, China) was used as the culture medium for ESCC cell lines. HEK293T was cultured in Dulbecco's modified Eagle's medium (DMEM).
Reagents and antibodies
Nobiletin (CAS#478-01-3) was purchased from Shanghai Rechemscience (Shanghai, China). Z-VAD-FMK (Cat#HY-16658B), 3-MA (Cat#HY-19312), and Fer-1 (Cat#HY-100579) were obtained from MedChemExpress (Shanghai, China). Erastin (Cat#GC6630) was purchased from Glpbio Technology (Montclair, CA, United States). Puromycin dihydrochloride (Cat#IP1280) was obtained from Solarbio (Beijing, China). LipofectamineTM 2000 (Cat#11668019) was purchased from Invitrogen (Shanghai, China). Anti-ZIP8 (Cat#20459-1-AP), anti-GPX4 (Cat#14432-1-AP), and anti-Beta Actin antibodies were obtained from Proteintech Group (Chicago, IL, United States). Anti-Phospho-CREB (Ser133) (Cat#9198) and anti-CREB (Cat#9197) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-FTL (CAS#ab109373) and anti-FTH1 (CAS#ab183781) antibodies were acquired from Abcam (Cambridge, UK). 666 − 15 (GC32689) was purchased from GLPBIO (Montclair, CA, America).
Cell counting kit-8 (CCK-8) assay
A CCK-8 kit (Meilunbio, Dalian, China) was utilized following the provided instructions to assess cell viability. Briefly, cell lines were seeded into a 96-well plate at a density of 1000 cells per well and treated with different concentrations of Nobiletin. The plate was then incubated for 24 to 96 hours at 37°C. Afterward, the culture medium was replaced with CCK-8 working solution containing 10% CCK-8 reagent. The cells were further cultured at 37°C for 1 hour. Finally, the absorbance at 450 nm in each well was measured using a microplate reader.
Colony formation assay
Cell lines were seeded into a 6-well plate at a density of 400 cells per well, followed by treatment with different concentrations of nobiletin. The plates were then incubated at 37℃ in a 5% CO2 environment. Once visible colonies became apparent in the culture dish, cultivation was stopped. The culture medium was discarded, and the cells were gently washed 2–3 times with PBS. Subsequently, 1 mL of methanol was added to each well for fixation, and the fixing solution was discarded after 15 minutes. To stain the colonies, 1 mL of 0.1% crystal violet or Giemsa staining solution was added to each well, and the plates were stained for 10–30 minutes. After staining, the staining solution was slowly rinsed off with running water, and the plates were air-dried. Finally, the number of colonies containing more than 50 cells was counted under a microscope, and photographs were taken for documentation.
Anchorage independent cell growth assay
ESCC cell lines KYSE30, KYSE450, and KYSE510 were cultured in appropriate growth medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% CO2. A solution of 0.6% agarose was prepared by dissolving agarose powder in sterile distilled water or cell culture medium. The agarose solution was autoclaved to sterilize and then cooled to approximately 40–45°C. ESCC cells were detached using trypsin-EDTA solution and counted using a hemocytometer. An appropriate number of cells were suspended in complete growth medium. The cell suspension was added onto the solidified agarose-coated plates. Plates were gently swirled to ensure uniform distribution of cells and then placed back into the incubator. Cells were allowed to grow for 2 weeks, with regular monitoring under a phase-contrast microscope.
Immunohistochemistry Analysis
The ESCC tissue array, obtained from Outdo Biotech (Shanghai, China), was subjected to immunohistochemistry (IHC) staining. The tissue array was initially dewaxed in xylene and then rehydrated using a series of alcohol concentrations (100%, 95%, 75%, and 50%). Following three washes with TBST, an antigen retrieval step was performed using 10 mM citrate buffer (pH 6.0) to expose the epitopes. To block endogenous peroxidase activity, the tissues were treated with 3% hydrogen peroxide for 5 minutes at room temperature. Subsequently, the tissues were incubated overnight at 4℃ with a primary antibody against ZIP8 (1:100 dilution). Afterward, the tissues were incubated with an HRP-IgG secondary antibody at 37℃ for 15 minutes. Diaminobenzidine staining was then performed for 2 minutes, followed by counterstaining with hematoxylin. Representative fields on each slide were captured using an inverted microscope, and the obtained images were quantified using Image-Pro Plus software.
Cell sample preparation and proteomics analysis
KYSE450 cells (1 × 106) were treated with 40µM nobiletin for 24 hours, after which the cells were collected for protein extraction. Digestion of protein (200µg for each sample) was performed according to the FASP procedure [26]. Peptides were labeled with TMT reagents according to the manufacturer’s instructions (Thermo Fisher Scientific). TMT-labeled peptides mixture was fractionated using a Waters XBridge BEH130 column (C18, 3.5 µm, 2.1 × 150 mm) on a Agilent 1290 HPLC operating at 0.3 mL/min. A total of 30 fractions were collected for each peptides mixture, and then concatenated to 15 (pooling equal interval RPLC fractions). The fractions were dried for nano LC-MS/MS analysis. LC-MS analysis was performed on a Q Exactive mass spectrometer that was coupled to Easy nLC (Thermo Fisher Scientific). The resulting LC-MS/MS raw files were imported into Proteome Discoverer 2.4 software (version 1.6.0.16) for data interpretation and protein identification against the database.
Computer docking model
PubChem served as the basis for the three-dimensional (3D) nobiletin structure, and ZIP8 structure derived from PDB database. AutoDockTools-1.5.7 software was used to simulate docking, and select the genetic algorithm parameters. Pymol was used to prepare and analysis molecular docking.
Drug affinity responsive target stability (DARTS) assay
The drug affinity responsive target stability (DARTS) method was performed following a procedure previously described [23]. Cells were collected and lysed with lysis buffer containing protease inhibitor and phosphatase inhibitors. The lysis buffer was then supplemented with TNC buffer. The lysates were aliquoted into 1.5 ml tubes and then incubated with different concentration of tangeretin or DMSO for 1h at room temperature. For exactly 30 min, the lysates were digested with 1µg of pronase for every 300 µg of lysate. Then, protein loading buffer was immediately added, the lysates were then heated to stop proteolysis. Western blotting was performed as previously described.
Cellular thermal shift assay (CETSA) assay
The cellular thermal shift assay (CETSA) was performed as described previously. Cells were incubated with 3 µM tangeretin or DMSO for 1h and washed with PBS three times. The cells were then suspended in 1ml PBS and divided into equal volumes, followed by heating at the indicated temperatures for 3 min. To lyse the cells, three snap freeze-thaw cycles were applied after heating. Western blot analysis was performed for further analysis.
Solvent-induced protein precipitation (SIP) assay
For the SIP assay, ESCC cells were lysed and incubated with DMSO or nobiletin. The five replications were treated with an organic solvent mixture comprising acetone: ethanol: acetic acid at a ratio of 50: 50: 0.1 to reach a final solvent concentration of 16%. Subsequently, the mixtures were equilibrated at 800 rpm for 20 min at 37°C. The supernatants were used for Western-blotting analysis.
Western-blotting analysis
Whole cell lysates were prepared by treating cells with RIPA buffer supplemented with a protease inhibitor (MCE). The cellular extracts were then centrifuged at 13,000 g for 10 minutes at 4℃. The protein concentration in the supernatant was determined using a BCA protein assay kit (PIERCE). A total of 20 mg of cell lysates were boiled for 10 minutes and separated by SDS-PAGE. The proteins were then transferred onto a PVDF membrane. Standard western blotting procedures were carried out using primary antibodies against ZIP8 (Santa Cruz), CREB (proteintech), pCREB (proteintech), GPX4 (proteintech), FTL (proteintech), and FTH1 (proteintech). The membrane was subsequently incubated with an HRP-conjugated secondary antibody (Santa Cruz) for 1 hour. After washing with PBST, the protein bands were detected by using the Western Bright ECL reagent (Advansta) and visualized with a ChemiDoc Touch Imaging System (Bio-Rad).
Plasmid Construction, Transfection, and Lenti-Virus Transduction
KYSE30, KYSE450, and KYSE510 cell lines were transfected with short hairpin RNA (shRNA) targeting ZIP8. The shZIP8 sequences were cloned into the lentiviral expression vector plko.1. The specific shZIP8 sequences used in this study were as follows: shZIP3#: 5'-CCG GCC TTG CTA TTC AAC TTC CTT TCT CGA GAA AGG AAG TTG AAT AGC AAG GTT TTT G-3' shZIP4#: 5'-CCG GGC TGC ACT TCA ACC AGT GTT TCT CGA GAA ACA CTG GTT GAA GTG CAG CTT TTT G-3'. The transfection process was performed following a previously established protocol (reference to previous study) [27]. The efficiency of transduction was assessed by Western blot analysis, confirming the knockdown of ZIP8 protein expression. Cell growth and colony formation ability were compared between the shZIP8-transfected cells and mock-transfected cells to evaluate the impact of ZIP8 knockdown on these cellular processes. ZIP8 overexpressing vector was purchased from Youbao Bio.
MDA measurement
Intracellular concentrations of malondialdehyde (MDA) were determined using a Cellular Lipid Peroxidation MDA Assay Kit (Cat#A003-1, Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). Following the indicated treatments, ESCC cells were harvested. The measurement of MDA was conducted in accordance with the manufacturer's instructions provided with the assay kit.
Erastin Sensitivity Assay
Seed cells in plates and reach optimal growth conditions. Prepare a stock solution of Erastin in a suitable solvent according to manufacturer instructions. Add the Erastin solution to the culture medium at different concentrations (0 µM, 0.01 µM, 0.1 µM, 1 µM, 10 µM) to create a dose-response curve. Incubate the cells with Erastin for a specified time period 24 hours. The viability of the cells was evaluated using CCK-8.
FerroOrange assay
Prepare the FerroOrange staining solution by following the manufacturer's instructions. Remove the culture medium from the treated cells and wash them gently with warm phosphate-buffered saline (PBS). Add the prepared FerroOrange staining solution to each well or plate containing the cells. Incubate the cells with FerroOrange staining solution for 30 minutes at room temperature, protected from light. Wash the cells again with warm PBS to remove excess stain. Visualize the stained cells using fluorescence microscopy equipped with appropriate filters for the excitation and emission wavelengths of FerroOrange.
C11-BODIPY assay
Cells were incubated for 48 hours under standard culture conditions. Prepare the C11-BODIPY staining solution by following the manufacturer's instructions. Remove the culture medium from the treated cells and wash them gently with warm phosphate-buffered saline (PBS). Add the prepared C11-BODIPY staining solution to each plate containing the cells. Incubate the cells with C11-BODIPY staining solution for 30 minutes at room temperature, protected from light. Wash the cells again with warm PBS to remove excess stain. Visualize the stained cells using fluorescence microscopy or a fluorescence plate reader equipped with appropriate filters for the excitation and emission wavelengths of C11-BODIPY.
Patient-derived xenograft (PDX) mouse model
The research protocol was granted approval by the Zhengzhou University Institutional Animal Care and Use Committee located in Zhengzhou, Henan, China. We procured six-week-old female SCID mice from Vital River Labs in Beijing, China, and housed them under a 12/12 hour light/dark cycle, providing them with unrestricted access to food and water. We dissected PDX tumor tissue into 1–2 mm fragments and transplanted these into the right flanks of the mice. To reduce variability in our experiments, we selected 10 mice for each group. Once the average tumor volume reached approximately 100 mm3, the mice were allocated randomly into two groups, each comprising 10 mice: one group received a vehicle control (0.9% saline), the second group was treated with nobiletin at a dose of 30 mg/kg, and the third group was treated with nobiletin at a dose of 60 mg/kg. We monitored tumor volume and body weight twice weekly, calculating tumor volume. Upon reaching a tumor volume of 800 mm3, the mice were euthanized, and the tumors were harvested. A portion of the tumors was preserved in formalin for hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) analysis, while the remainder was stored at -80℃ for subsequent protein analysis.
Statistical analyses
SPSS 23.0 was used for statistical analyses in this study, and quantitative data were presented as the mean ± SD. To compare significant differences, the one-way analysis of variance (ANOVA) or unpaired Student’s t-test was used. For each experiment, *p < 0.05, **p < 0.01, and ***p < 0.001 were chosen to indicate significance.