Cell culture

HepG2 and SMMC7721 Cells were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The cells were cultured in DMEM (Gibco, #11960-044) supplemented with 10% fetal Bovine serum (Gibco, #10099141) and 1% penicillin and streptomycin (Gibco, #15070-063). All the cells were maintained with 5% CO2 in a 37 °C incubator.

In vitro cell proliferation assays

The abilities of proliferation of HCC cells in vitro were evaluated by Cell Counting Kit-8 (CCK-8), colony formation, LDH assay, and Calcein/PI assay staining.

For the CCK-8 assay, cells were inoculated into 96-well plates at a density of 3000 cells/well. CCK-8 Reagent (Epizyme Biotechnology Co., Ltd, #CX001) was used to assess cell viability. Briefly, A cell suspension with a cell density of 5000 cells per well was prepared and incubated in a 96 well plate for a specific period. Add 10 μl of CCK8 solution to each well and allow the cck8 reagent to react with viable cells for 4 h. Measure the absorbance of the pores using a microplate reader at a wavelength of 450 nm. The cell viability is calculated by comparing the absorbance values of the treated cells with those of the control cells. Absorbance was measured at 450 nm using an enzyme meter.

For colony formation assay, cells were inoculated in 6-well plates at a density of 100 cells per well and incubated for two weeks in complete medium with different treatment factors. The growing colonies were stained with crystal violet staining reagent (Beyotime Technology Co., Ltd.#C0121). Images were taken with a smartphone and clearly visible colonies (> 20 cells/colony) were counted. The plate clone formation experiment was carried out according to the following steps. A cell suspension with a cell density of 1000 cells per well was cultured in a 6-well plate for 48 h, and corresponding drugs were added for intervention to form colonies. After this, fix and use crystal violet staining solution for cell colony staining. A microscope was used for counting and analyze the form colonies.

Lactate dehydrogenase (LDH)Cytotoxicity Assay Kit (Beyotime Technology Co., Ltd.#C0017), Inoculate the appropriate amount of cells into a 96-well cell culture well plate according to the size and growth rate of the cells, so that the density of cells at the time of the test to be performed does not exceed 80–90% full. Different drugs were added for treatment and appropriate controls were set up. After drug stimulation, the cell culture plate was centrifuged with a multiwell plate centrifuge at 400 g for 5 min. Aspirate the supernatant as much as possible, add 150 μl of LDH release reagent provided in the kit diluted tenfold with PBS (1 volume of LDH release reagent was added to 10 volumes of PBS and mixed, and shake the plate appropriately to mix well and then continue to incubate the plate for 1 h in the cell culture incubator. Subsequently, the cell culture plates were centrifuged in a multiwell plate centrifuge at 400 g for 5 min. 120 μl of the supernatant from each well was taken separately and added to the corresponding well of a new 96-well plate, followed by sample determination and the absorbance of the samples was detected at 490 nm with an enzyme marker.

Cell migration in vitro

The ability of HCC cells to metastasize in vitro was assessed by the wound scratch test. Cells grown to the filling stage were scratched with a 100 μl pipette tip and incubated in serum-free medium for 12, 24, and 48 h. Images were taken at the beginning and end of the experiment at 100 × magnification.

For Calcein/PI assay (Beyotime Technology Co., Ltd.#C2015), Adherent cells were trypsin-digested and resuspended in culture medium and washed once with PBS; suspended cells were centrifuged at 250–1000 × g for 5 min at room temperature, the supernatant was discarded and washed once with PBS. The recommended cell dosage for each sample was 106 cells. For the precipitate of 106 cells from the previous step, add 1 ml of Calcein AM/PI assay working solution and resuspend to a single-cell suspension. incubate for 30 min at 37 °C, protected from light. note: It is necessary to prepare a buffer-only cell sample to be used as a negative control for the flow cytometry assay, which is suitable to maintain the same buffer as that used to prepare the Calcein AM/PI assay working solution. Two additional tubes of cell samples are also prepared with only one dye (Calcein AM or PI) added to each tube for compensatory adjustment of the flow single stain. After incubation, the cells can be directly detected by flow cytometry, or centrifuged at 250–1000 × g for 5 min at room temperature to precipitate the cells, and after aspirating the liquid, 0.5 ml of buffer was added to each sample to resuspend the cells and then detected by flow cytometry.

Protein isolation and Western blot analysis

Proteins were extracted using RIPA reagent (Epizyme Biotechnology Co., Ltd, #PC103) and protein concentrations were quantified using a BCA reaction kit (Beyotime Technology Co., Ltd.#P0011). Equal amounts of proteins (25-50 μg) were separated by vertical electrophoresis on 10% SDS-PAGE (Beyotime Technology Co., Ltd.#P0015) and then electro transferred onto a PVDF membrane (Thermo-Fisher Scientific, Pittsburgh, PA, USA). The PVDF membranes were incubated in a protein-free rapid containment solution for 15 min, and then the membranes were incubated with primary antibodies ACSL4 (1: 10,000; ABclonal Biotechnology, China#A20414), PTGS2 (1:1000; ABclonal Biotechnology, China#A3560), GPX4 (1: 1000; ABclonal Biotechnology, China#A11243), SLC7A11 (1: 1000; ABclonal Biotechnology, China#), FTH1 (1: 1000; ABclonal Biotechnology, China#A11243), GSS (1: 500. ABclonal Biotechnology, China#A11557), GAPDH (1: 10,000; ABclonal Biotechnology, China#A19056) at 4° overnight. The next day, HRP-labeled secondary antibody (1: 10,000; ABclonal Biotechnology, China# AS014) was added and developed by chemiluminescence with ECL reagent (Epizyme Biotechnology Co., Ltd, # SQ201). And the expression levels of target proteins were analyzed semi-quantitatively by Image J.

transient transfection

Cells were seeded at 8000 per well in 6-well plates, and the shACSL4 plasmid was transfected using Poly plus transfection reagent, and the medium was replaced with fresh complete medium after 12 h of transfection for subsequent experiments.

Reagents

Curcumin(HY-N0005, Purity: 98.16%), Ferrostatin-1(HY-100579), Liproxstatin-1 (HY-12726),Z-VAD-FMK (HY-16658B), Necrostatin-1(HY-15760), 3-Methyladenine(HY-19312), Deferoxamine mesylate (HY-B0988) were purchased from MedChemExpress.

Malondialdehyde (MDA) and glutathione (GSH) assay

Cells were seeded in cell culture plates at 8000 cells per well, and after 24 h of incubation, cell lysates were collected for detection of intracellular MDA (Beyotime Technology Co., Ltd.#S0131) and GSH (Beyotime Technology Co., Ltd.#S0052) content according to the kit instructions. MDA was detected at 535 nm and GSH at 412 nm with an enzyme marker.

Iron assay

For cells, cellular ferrous ion levels were detected with the ferroOrange probe (Dojindo#F374). Cells were seeded at 8000 per well in a six-well plate, cell crawls were made, and after 24 h, the probe was stained for 30 min and washed three times with PBS. Subsequently, the cellular ferrous ion level was observed by laser confocal microscopy. For tissues, 100 mg of tumor tissue was lysed and the supernatant was taken after centrifugation at 12,000 g. Reaction reagents were added sequentially according to the instructions. Add all Standard tubes, sample tubes, and sample blank tubes to a 96-well plate and incubate for 60 min, then measure the absorbance at 593 nm. The total and ferrous iron content of the tissue was also calculated according to the standard curve.

Hydrogen peroxide and superoxide detection assay

Intracellular hydrogen peroxide and superoxide levels were used to evaluate the level of intracellular oxidation.

For the hydrogen peroxide assay, cells were seeded in 96-well plates and after 24 h of drug incubation, supernatants were taken and assayed according to the assay kit instructions (Nanjing Jiancheng Bioengineering Institute#A064-1-1).

For the superoxide assay (Beyotime Technology Co., Ltd.#S0060), cells were seeded in 96-well plates and drug interventions were added for 24 h, then the superoxide kit instructions were followed, and the absorbance of the samples was read at 450 nm.

Mitochondrial membrane potential assay

Cells were seeded at 5000 per well in 12-well plates, and after different groups were given different drugs for 24 h of stimulation, intracellular mitochondria were localized and stained using Mito-Tracker Red CMXRos (Beyotime Technology Co., Ltd.#S0131), and the mitochondrial membrane potential was observed under an inverted fluorescence microscope, and the average fluorescence intensity was calculated using Image J. The cells were then stained with Mito-Tracker Red CMXRos, and the mitochondrial membrane potential was measured using Mito-Tracker Red CMXRos.

Immunohistochemistry

The immunohistochemistry (IHC) procedure involves several steps. First, the tumor tissues were fixed and embedded. Then, it is cut into thin sections measuring 4 μm. Antigen repair was performed in sodium citrate buffer at 95–100 °C for 40 min. To minimize non-specific binding, 5% BSA was used for blocking. The primary antibody was incubated at 4 °C overnight to facilitate specific binding of the target antigen. Subsequently, the secondary antibody, conjugated with a detection molecule, was incubated to bind to the primary antibody. The visualization of target proteins was achieved through the utilization of DAB colorimetric solution. Subsequently, a quantitative analysis of each sample was conducted from three distinct perspectives.

Lipid peroxidation assay

Cells were seeded in six-well plates at 8000 per well, and after 24 h, different groups of drugs were given to stimulate for 24 h, liperfluo probe (Dojindo#L248) was incubated for 30 min, PBS washed three times, and then cells were digested by 0.25% EDTA trypsin in flow tubes and detected by flow cytometer on the machine.

Animal model

To establish a mouse model of hepatocellular carcinoma xenografts, HepG2 cells were injected subcutaneously into the right abdomen of 5- to 6-week-old nude mice. Approximately 107 cells were injected into each. The maximum width (X) and length (Y) of the tumors were measured with calipers every three days, and the volume (V) was calculated using the following equation. The volume (V) was calculated using the formula: V = (X2Y)/2. The mice were randomly divided into two groups: (1) a drug group, in which the mice were injected intraperitoneally with curcumin (20 mg/kg) every day and fed a normal diet; and (2) a control group, in which the mice were injected intraperitoneally with an equal amount of saline and fed a normal diet every day. mice were executed for material collection after 30 days.

All animal experimental protocols were approved by the Animal Center and Use Committee of Shanghai University of Traditional Chinese Medicine.

Statistical analysis

All data was shown as mean and Standard Deviation (SD). The Kolmogorov–Smirnov or Shapiro–Wilk test was used to determine if the data were regularly distributed. Comparisons of the significance between two variables assuming they were regularly distributed aired or unpaired two-tailed T tests were used to analyze the groups, and Comparisons of three or more groups' importance were made either a two- or one-way ANOVA. If the distribution of the data were skewed, utilizing comparisons of the importance between two groups, comparisons of significance using the nonparametric Mann–Whitney U test utilizing the Kruskal–Wallis test across three or more groups.