Mouse embryonic fibroblasts (MEFs) (CRL-2991; ATCC) and the HT1080, DU145 and H1299 cell lines were obtained from the American Tissue Culture Collection (ATCC) and cultured following ATCC recommendations. These cells were also subjected to STR identification and mycoplasma testing. The cells were sustained in Dulbecco's modified Eagle's medium (DMEM) with high glucose, sodium pyruvate (1 mM), glutamine (2 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml) and 10% (v/v) fetal bovine serum (FBS; MeisenCTCC; Zhejiang Meisen Cell Technology Co., Ltd.) at 37°C and 5% CO₂. In addition, the cultivation conditions for methionine deficiency are based on DMEM (cat. no. D0422; MilliporeSigma) with high glucose, sodium pyruvate (1 mM), glutamine (2 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml) and 10% (v/v) fetal bovine serum (FBS) (MeisenCTCC) at 37°C and 5% CO₂. Alternatively, S-adenosylmethionine (SAM; 0.4 mM), S-adenosylhomocysteine (SAH; 0.4 mM), homocysteine (Hcy; 0.4 mM) or adenosine dialdehyde (ADOX; 0.4 mM) were added separately to methionine deficient culture medium to treat cells. Cells were also treated with a constructed culture medium lacking essential amino acids (isoleucine, 0.4 mM; isoleucine, 0.4 mM; lysine, 0.4 mM; methionine, 0.4 mM; phenylalanine, 0.4 mM; threonine, 0.4 mM; tryptophan, 0.4 mM; valine, 0.4 mM; histidine, 0.4 mM; and arginine, 0.4 mM).

The reagents used included propidium iodide (PI; cat. no. P4170; MilliporeSigma), BODIPY581/591 C11 (cat. no. D3861; Thermo Fisher Scientific, Inc.), BODIPY (cat. no. HY-D1614; MedChemExpress), BODIPY^™ FL C12 (cat. no. D3822; Thermo Fisher Scientific, Inc.), SAM (cat. no. A7007; MilliporeSigma), S-Adenosylhomocysteine (SAH; cat. no. A9384; MilliporeSigma), homocysteine (cat. no. H4628; MilliporeSigma), anti-MAT2A (cat. no. A19272; ABclonal Biotech Co., Ltd.), anti-ACSL4 (cat. no. A20414; ABclonal Biotech Co., Ltd.) and DMEM with high glucose (cat. no. D0422-100 ml; MilliporeSigma). In addition, all the components of the D777 culture medium (with 4,500 mg/l glucose, L-glutamine and sodium pyruvate, without sodium bicarbonate, powder, suitable for cell culture) were purchased to construct an amino acid-deficient culture medium. The medium mainly included methionine (cat. no. M0960000), leucine (cat. no. L8000), isoleucine (cat. no. I2752), lysine (cat. no. L5501), phenylalanine (cat. no. P17008), tyrosine (cat. no. T1145), tryptophan (cat. no. T0254), valine (cat. no. V0500), histidine (cat. no. H8125) and arginine (cat. no. A5006), all from MilliporeSigma.

Fatty acid uptake was measured by flow cytometry. Briefly, HT1080 and H1299 cells were plated at a density of 5×10⁴ cells per well in a 24-well dish and cultured for 12 h in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin at 37°C with 5% CO₂. Subsequently, 2 µM BODIPY-C12 was added to the cell culture medium and incubated for 10 h alongside the indicated treatment. Following this, the cells were trypsinized, washed with PBS containing 5% FBS and resuspended in 0.3 ml of PBS with 5% FBS for flow cytometry analysis (Attune NxT; Thermo Fisher Scientific, Inc.). Acquired data were analyzed using FlowJo version 10 software (FlowJo LLC). A minimum of 10,000 cells were analyzed per condition. Intracellular lipids were also measured by flow cytometry (Attune NxT.). Acquired data were analyzed using FlowJo version 10 software. The protocol was the same as that aforementioned except 1 µg/ml BODIPY was added to the cell culture medium and incubated for 30 min after the indicated treatment.

Lipid ROS were analyzed by flow cytometry. Briefly, cells were plated at a density of 1×10⁵ cells per well in a 24-well dish and cultured overnight in DMEM. Subsequently, 4 µM BODIPY581/591 C11 was added to the cell culture medium and incubated for 30 min after the indicated treatment. Excess BODIPY581/591 C11 was then removed by washing the cells with PBS twice. Labeled cells were trypsinized and resuspended in PBS with 5% FBS. Oxidation of BODIPY581/591 C11 resulted in a shift of the fluorescence emission peak from 590 to 510 nm proportional to the lipid ROS generation, which was detected using a flow cytometer (Attune NxT). Acquired data were analyzed using FlowJo version 10 software.

Cell death was analyzed by flow cytometry. Briefly, cells were plated at a density of 2×10⁵ cells per well in a 12-well dish for 12 h in fresh medium. After the indicated treatment, 1 µg/ml PI was added to the cell culture medium and incubated for 30 min. Then, cells were trypsinized, washed with PBS containing 5% FBS and resuspended in 0.3 ml of PBS with 5% FBS for flow cytometry (Attune NxT). Acquired data were analyzed using FlowJo software.

Apoptosis was analyzed by flow cytometry. Briefly, cells were plated in 12-well culture plates at a density of 1×10⁵ cells per well and cultured in DMEM for 12 h. Cells were then trypsinized, washed with PBS containing 5% FBS and resuspended in 0.3 ml of PBS. Subsequently, PI and Annexin V-FITC were added to the cells and incubated for 30 min, and cell apoptosis was detected by flow cytometry (Attune NxT). Acquired data were analyzed using FlowJo.

Transfections were conducted using Lipofectamine^™ 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. HT1080 cells seeded in a 6-well dish were transfected with 40 ng of the following small interfering RNAs (Suzhou GenePharma Co., Ltd.): NC (scrambled), sense: 5′-UUCUCCGAACGUGUCACGUUTT-3′, antisense: 5′-AACGUGACACGUUCGGAGAATT-3′; MAT2A-1, sense: 5′-GGAUCGAGGUGCUGUGCUUTT-3′, antisense: 5′AAGCACAGCACCUCGAUCCTT-3′; MAT2A-2, sense: 5′-GGGAUGCCCUAAAGGAGAATT-3′, antisense: 5′-UUCUCCUUUAGGGCAUCCCTT-3′ and MAT2A-3, sense: 5′-GCCUAUGGCCACUUUGGUATT-3′, antisense: 5′-UACCAAAAGUGGCCAUAGGCTT-3′. After transfection, the cells were placed in a 37°C cell culture incubator and the medium was changed after 8 h. After 48 h, the cells were lysed and the interference efficiency was measured using western blotting.

Cell lysate preparation, SDS-PAGE and electrophoretic transference were accomplished as previously described (19). Briefly, the protein sample was quantified using a NanoPhotometer^® N30 Touch and each lane of the 8% SDS gel was loaded with 10 µg protein sample. Membranes were blocked with 5% non-fat dried milk in TBS (pH 7.2) containing 0.1% Tween 20 (TBST), incubated with the appropriate primary antibodies in 5% non-fat dried milk in TBST overnight at 4°C. Then, the membrane was incubated with the appropriate secondary antibodies in 5% non-fat dried milk in TBST at room temperature for 50 min. The following primary antibodies were used: Anti-MAT2A, anti-ACSL4 and β-actin (cat. no. HC201-01; TransGen Biotech Co., Ltd.). The following secondary antibodies were used: HRP-conjugated goat anti-rabbit (cat. no. AS014; ABclonal Biotech Co., Ltd.) and HRP-conjugated goat anti-mouse (cat. no. AS003; ABclonal Biotech Co., Ltd.). Chemiluminescence was detected using the ChampChemi 610 (SinSage Technology Co., Ltd.), and the software used for densitometry was ImageJ (National Institutes of Health).

Total RNA was extracted from cells using TRNzol (cat. no. DP424; Tiangen Biotech Co., Ltd.) and reverse transcribed with the PrimeScript^™ RT reagent Kit with gDNA Eraser (cat. no. RR047A; Takara Bio, Inc.) according to the manufacturer's protocol. The resulting cDNA was then subjected to qPCR using the TB Green Premix Ex Taq^™ (Tli RNase H Plus) (cat. no. RR820A; Takara Bio, Inc.). The detection and analysis were accomplished as previously described (19). The sequences of the primers used for qPCR were as follows: ACSL4 (human) forward, 5′-CATCCCTGGAGCAGATACTCT-3′ and reverse, 5′-TCACTTAGGATTTCCCTGGTCC-3′; MAT2A (human) forward, 5′-ACCAGAAAGTGGTTCGTGAAG-3′ and reverse, 5′-CAAGGCTACCAGCACGTTACA-3′; MAT2B (human) forward, 5′-TTCACTGGTCTGGCAATGAAC-3′ and reverse, 5′-AGGGCTGTCAGTAATAGGTCTT-3′; MAT2A (mouse) forward, 5′-GCTTCCACGAGGCGTTCAT-3′ and reverse, 5′-AGCATCACTGATTTGGTCACAA-3′; MAT2B (mouse) forward, 5′-AGGGAACCTTTCACTGGTCTG-3′ and reverse 5′-ATTTGGAGCAATCGAGCTGAG-3′; β-actin (human) forward, 5′-GTTGTCGACGACGAGCG-3′ and reverse, 5′-GCACAGAGCCTCGCCTT-3′; and β-actin (mouse) forward, 5′-GGCTGTATTCCCCTCCATCG-3′ and reverse, 5′-CCAGTTGGTAACAATGCCATGT-3′.

The survival analysis module on the GEPIA 2 website was used to analyze the relationship between ACSL4 expression and patient survival. The analysis parameter settings were as follows: Methods (overall survival); group cut-off (median); cut-off high (%) (50); cut-off low (%) (50); hazard ratio (yes); 95% confidence interval (yes); and axis units (months).

GraphPad Prism (v.10.4.0; Dotmatics) software was used to calculate the P-values using two-sided unpaired t-test or one-way ANOVA followed by Tukey's Honestly Significant Difference. All data presented in the figures represent the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

To systematically evaluate the role of essential amino acids in lipid metabolism regulation, an essential amino acid-deficient culture system was established by excluding leucine, isoleucine, lysine, methionine, phenylalanine, tyrosine, tryptophan, valine, histidine and arginine from the medium. HT1080 cells cultured in this modified medium were subsequently analyzed for intracellular lipid accumulation using BODIPY staining. Quantitative analysis revealed that methionine deprivation specifically caused a significant reduction in cellular lipid content (Fig. 1A). Next, the functional consequences of essential amino acid deficiency on fatty acid uptake capacity were examined using BODIPY-C12 fluorescence assays. Notably, methionine-deficient conditions produced a notable impairment in fatty acid internalization among all tested amino acid deprivations (Fig. 1B).

Given the established role of ACSL4 in facilitating fatty acid uptake through its dual function of converting fatty acids to acyl-CoA derivatives and inhibiting fatty acid efflux, ACSL4 protein expression in HT1080 cells under essential amino acid deprivation conditions was first examined. Western blot analysis revealed that methionine deficiency reduced the ACSL4 protein levels (Fig. 1C). To validate this finding across different cellular contexts, the investigation was extended to MEFs under methionine-restricted conditions. Consistent with the initial observation, methionine deprivation significantly decreased ACSL4 expression in MEFs cells (Fig. 1D). Taken together, the results suggest that methionine deficiency attenuates cellular lipid accumulation and the fatty acid uptake capacity, potentially through downregulation of ACSL4 protein expression, revealing a novel mechanistic link between methionine availability and lipid metabolic regulation.

As the sulfur-containing amino acid methionine functions as a vital cellular methyl donor primarily through its metabolic derivative SAM, it was next determined whether methionine regulates ACSL4 expression and fatty acid uptake capacity via SAM-dependent mechanisms. To test this hypothesis, methionine deprivation experiments in HT1080 cells were performed followed by SAM supplementation. Western blot analysis demonstrated that SAM administration rescued the methionine deficiency-induced reduction in ACSL4 protein levels (Fig. 2A). Complementary RT-qPCR analysis further revealed that SAM supplementation similarly restored the suppressed ACSL4 mRNA expression under methionine-depleted conditions (Fig. 2B). To elucidate whether SAM regulates ACSL4 expression through methylation-dependent mechanisms, HT1080 cells were treated with the methylation inhibitor, adenosine dialdehyde (ADOX), and the ACSL4 transcript levels were analyzed. RT-qPCR analysis revealed that ADOX treatment significantly suppressed ACSL4 mRNA expression (Fig. 2C), indicating methylation-dependent regulation. This regulatory mechanism in H1299 cells was further validated, as western blot analysis confirmed that the methionine-SAM axis controls ACSL4 protein abundance possibly through methylation modifications (Fig. 2D). Functional assays demonstrated that this methylation-dependent regulation directly impacts cellular fatty acid uptake capacity (Fig. 2E). Collectively, these results establish that SAM-mediated methylation may represent a critical epigenetic mechanism through which methionine regulates ACSL4 expression and function in lipid metabolism.

As the key enzyme catalyzing methionine-to-SAM conversion, MAT2A occupies a central position in the methionine-SAM metabolic axis (24). To investigate whether MAT2A serves as a critical regulator of SAM homeostasis and lipid metabolism, MAT2A protein expression in HT1080 cells under essential amino acid deprivation was first examined. Western blot analysis revealed that the MAT2A levels were specifically upregulated by methionine deficiency (Fig. 3A). Using a dose-response approach, it was demonstrated that MAT2A expression exhibits sensitivity to extracellular methionine concentrations in DU145 cells (Fig. 3B). Notably, SAM supplementation reversed the observed methionine deprivation-induced MAT2A upregulation (Fig. 3C), suggesting a feedback regulatory mechanism. Further mechanistic studies in H1299 cells showed that MAT2A protein levels remained unchanged following ADOX-mediated methylation inhibition, compared with the methionine deprivation only group (Fig. 3D). Consistent with this result, transcriptional analysis across multiple cell lines (MEFs, HT1080 and H1299) revealed that, while the methionine-SAM axis regulates MAT2A mRNA expression, this control occurs independently of the methyltransferase activity of SAM (Fig. 3E). Notably, the regulatory subunit, MAT2B, was unaffected by these metabolic perturbations (Fig. 3F). These results establish that MAT2A is subject to SAM-mediated feedback regulation through a novel methylation-independent mechanism, highlighting the sophisticated control of methionine metabolism.

To systematically validate the regulatory role of methionine in lipid metabolism, complementary experimental approaches were performed using HT1080 cells. First, methionine deprivation (using commercial methionine deficient medium) led to significant reductions in both intracellular lipid accumulation (BODIPY staining) and fatty acid uptake capacity (BODIPY-C12 assay) (Fig. 4A and B). To directly assess the involvement of MAT2A in these metabolic processes, RNA interference-mediated knockdown was employed. MAT2A downregulation similarly reduced the intracellular lipid content (Fig. 4C and D) and impaired fatty acid internalization (Fig. 4E), phenocopying the effects of methionine deprivation. These consistent findings across orthogonal experimental approaches establish MAT2A as a key metabolic regulator and potential therapeutic target for modulating cellular lipid homeostasis.

Lipids serve as fundamental structural components of cellular membranes and play a critical role in supporting the rapid proliferation of tumor cells. The aforementioned results demonstrated that methionine functions as a positive regulator of intracellular lipid homeostasis. Analysis of The Cancer Genome Atlas datasets revealed elevated ACSL4 expression in multiple malignancies, including esophageal carcinoma, liver hepatocellular carcinoma, pancreatic adenocarcinoma and stomach adenocarcinoma (Fig. 5A). Notably, high ACSL4 expression was significantly associated with poorer overall survival in patients with cancer, including cholangiocarcinoma, esophageal carcinoma and colon adenocarcinoma (Fig. 5B), suggesting its clinical relevance as a potential prognostic marker. Functional studies in HT1080 cells showed that methionine deprivation simultaneously suppressed ACSL4 expression (Fig. 5C) and impaired HT1080 cell proliferation (Fig. 5D), while not inducing ferroptosis (Fig. 5E and F); however, it has a certain inducing effect on apoptosis (Fig. 5G). Taken together, these results establish methionine as an essential metabolic requirement for tumor cell proliferation, operating through ACSL4-mediated lipid metabolic pathways.