Cell culture, cell transfection, and small chemical compounds

The CRC cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Yuuta, Shanghai, China) supplemented with 10% fetal bovine serum (ExCell Bio, Shanghai, China) and maintained at 37 °C in a humidified 5% CO2 atmosphere. These cell lines were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (CAS), Shanghai, China. For experiments involving serine and glycine deprivation, the cells were cultured in a specialized medium that, similar to DMEM, lacked only serine and glycine (Biosharp, Cat. No. BL301A, Shanghai, China), and also supplemented with 10% fetal bovine serum (ExCell Bio, Cat. No. FSD500, Shanghai, China) under the same conditions of temperature and CO2 concentration.

Transient transfection was performed using reagents from HighGene (Cat. No. RM09014, ABclonal, Wuhan, China). Small interfering RNAs (siRNAs) targeting FOXC1, along with plasmids overexpressing FOXC1, were sourced from Gene Pharma (Shanghai, China). Similarly, siRNAs directed against PHGDH, PSAT1, and PSPH were obtained from Genomeditech (Shanghai, China). Lentiviral vectors, LV-Control and LV-shFOXC1, were acquired from Genechem Biotechnology (Shanghai, China). After transduction, cell lines exhibiting stable gene expression were selected and cultured in the presence of puromycin.

5-Fluorouracil (Cat. No. 343922), L-Serine (Cat. No. S4311), BI-D1870 (Cat. No. 559286) and U0126 (Cat. No. 662009) were obtained from Sigma-Aldrich (St. Louis, MO, USA), and NCT-503(Cat. No. Targetmol4213), was obtained from MedChemExpress (Monmouth Junction, NJ, USA)0.4-Fluoro-L-2-phenylglycin was purchased from Tokyo Chemical Industry (Cat. No. F0862, Japan).

Western blot (WB) analysis

Proteins were extracted from cultured cells and quantified using a Bradford assay. The proteins were separated by SDS-PAGE, transferred onto PVDF membranes (Cat. No. 3010040001, Millipore Corporation, Burlington, MA, USA), and probed with specific antibodies. Antibodies recognizing, PSAT1(Cat. No. PA5-22124, RRID: AB_11153526, Thermofishe, 1:2000), PSPH (Cat. No. A22763, ABclonal, 1:2000), PHGDH (Cat. No. A22129, ABclonal, 1:2000), FOXC1(Cat. No. ab227977, RRID: AB_2916124, Abcam, 1:1000), ERK1/2 (Cat. No. ab184699, RRID: AB_2802136, Abcam, 1:1000), phospho-ERK1/2(Cat. No. ab201015, RRID: AB_2934088, Abcam, 1:1000), RSK1(Cat. No. A4695, RRID: AB_2863326, ABclonal, 1:2000), phospho-RSK1(Cat. No. AP0539, RRID: AB_2771393, ABclonal, 1:2000), ELK1 (Cat. No. A19046, RRID: AB_2862539, ABclonal, 1:2000), phospho-ELK1(Cat. No. AP0033, RRID: AB_2771079, ABclonal, 1:3000), γH2AX (Cat. No. AP0687, RRID: AB_2863808, ABclonal, 1:2000) and β-actin (Cat. No. AC026, RRID: AB_2768234, Abclonal,1:10000).

RT‑qPCR assays and bioinformatic analysis

Total RNA was extracted using TRIzol reagent (Cat. No. A33254, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The isolated RNAs were reverse transcribed into complementary DNAs using the PrimeScript RT reagent Kit (Code No. RR036A, Takara Bio, Tokyo, Japan). Quantitative real-time PCR (RT-qPCR) was subsequently performed using SYBR Green I (Cat. No. RR420A, Takara Bio). Primer sequences used are listed in Supplementary Table S1. Expression levels were normalized to β-actin as an internal control.

A comprehensive cancer genomics initiative, The Cancer Genome Atlas (TCGA), has provided molecular characterizations across 33 primary cancer types. For the analysis of FOXC1, ATF4, PHGDH, PSPH, and PSAT1 expression in colorectal cancer (CRC), data were extracted from the TCGA database and analyzed using the UALCAN platform (https://ualcan.path.uab.edu/analysis.html).

For RNA sequencing, SW1116 cells were cultured for 24 h in complete medium (Comp) or in a medium deprived of serine/glycine (Ser(-)). RNA was extracted, and library preparation for RNA-seq was conducted using 1 µg of high-quality RNA. Sequencing was performed on a HiSeq2500 platform (Illumina Inc., San Diego, CA, USA) at Genechem Biotechnology (Shanghai, China).

Serine and glycine measurement

Intracellular serine and glycine concentrations were quantified using the Serine Assay Kit and the Glycine Assay Kit, respectively, sourced from Mlbio (Cat. No. ml077310, Cat. No. Cat. No. ml077310, Shanghai, China). The assays were conducted with 100 µL of cell lysate in reaction buffer, prepared by the kit instructions. Fluorescence was subsequently measured in endpoint mode with a wavelength of 450 nm.

Cell counting Kit-8 (CCK-8) proliferation assay

To evaluate the proliferation of colorectal cancer (CRC) cells, Cell Counting Kit-8 (CCK-8) assays were conducted using kits provided by Dojindo Laboratories (Cat. No. CK04, Kumamoto, Japan). Briefly, CRC cells were seeded at a density of 2 × 10^3 cells per well in 96-well plates and cultured for durations of either 0–96 h or 0–144 h. Subsequently, each well received 10 µL of CCK-8 reagent mixed with 90 µL of DMEM. Following a one-hour incubation period, the absorbance at 450 nm was measured using a microplate reader. The experiments were performed in triplicate, and data analysis was conducted using GraphPad Prism software.

EdU Staining

The EdU incorporation assay was performed using the EdU Kit (Cat. No. ST067, Beyotime, Jiangsu, China) to assess cell proliferation. Cells were plated at a density of 5 × 10^5 cells per well in 24-well culture plates 24 h before the assay. After treatment with 50 µM EdU for 2 h, cells were fixed, permeabilized, and subsequently stained following the manufacturer’s instructions. The assay was conducted in triplicate with three independent experiments.

Intracellular GSH detection

Intracellular glutathione (GSH) levels were measured using a GSH and GSSG Assay Kit (Cat. No. S0053, Beyotime, Jiangsu, China). Cells were lysed through two cycles of freezing and thawing, followed by centrifugation to collect the supernatant. The quantification of GSH and GSSG was then conducted according to the manufacturer’s protocol.

Measurement of the NADPH/NADP + ratio and ROS levels

The NADPH/NADP + ratio was determined using a NADP/NADPH Quantification Colorimetric Kit (Cat. No. S0179, Beyotime, Jiangsu, China). For this assay, cell lysates were mixed with 800 µL of reaction buffer, prepared according to the kit instructions, and the absorbance was measured at 450 nm at room temperature in a 96-well plate. Additionally, reactive oxygen species (ROS) levels were quantified using a ROS Detection Assay Kit (Cat. No. S0033S, Beyotime, Jiangsu, China), with the procedure conducted as per the manufacturer’s guidelines.

Chromatin immunoprecipitation (ChIP) assay

The Chromatin Immunoprecipitation (ChIP) assay was performed using a kit from Upstate Biotechnology, provided by Thermo Fisher Scientific (Waltham, MA, USA). Cells were first cross-linked with 1% formaldehyde and 1.5 mmol/L ethylene glycol-bis at room temperature. The cross-linked chromatin was then sonicated and immunoprecipitated using antibodies specific to various transcription factors. Quantitative real-time PCR (qPCR) was conducted to quantify the amount of DNA bound, employing primers specifically designed to target the FOXC1 binding sites within the promoter regions of the PSAT1, PSPH, and PHGDH genes. Additionally, an experimental group targeting ELK1 binding to FOXC1 was included to explore interaction effects.

Immunohistochemistry (IHC)

Immunohistochemistry (IHC) was performed as follows: Tissue sections were fixed, embedded, sectioned, and deparaffinized. Blocking was achieved using 3% hydrogen peroxide and 5% bovine serum albumin (BSA). Subsequently, sections were incubated with primary antibodies against Ki-67(Cat. No. A11390, ABclonal, 1:200) and γH2AX(Cat. No. AP0687, ABclonal, 1:200) overnight at 4 °C. After washing with phosphate-buffered saline (PBS), sections were treated with an immunohistochemical secondary antibody for 1 h at room temperature. This was followed by diaminobenzidine (DAB) staining, counterstaining with hematoxylin, and imaging.

Apoptosis assay

Apoptosis was assessed using Annexin V-FITC/PI staining followed by flow cytometry analysis. Cells were harvested, washed twice with cold phosphate-buffered saline (PBS), and resuspended in 1x binding buffer at a concentration of 1 × 10^6 cells per mL. Annexin V-FITC (1:100) and propidium iodide (PI) (1:100) from Dojindo (Kumamoto, Japan) were added to the cell suspension, followed by incubation for 15 min at room temperature in the dark. After incubation, 400 µL of 1x binding buffer was added, and apoptosis was analyzed immediately by flow cytometry.The percentage of apoptotic cells was determined by gating for early apoptotic cells (Annexin V-FITC+/PI-) and late apoptotic/necrotic cells (Annexin V-FITC+/PI+). Flow cytometric data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

53BP1 foci formation assay

Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100, and blocked with 5% normal goat serum for 1 h. Cells were then incubated overnight with a primary antibody against 53BP1 (1:500, Abcam, Cat. No. ab175933) at 4 °C. After washing, cells were incubated with an Alexa Fluor 568-conjugated secondary antibody (1:1000, Thermofisher, Cat. No A-11004) for 1 h in the dark, and nuclei were stained with DAPI (1 µg/mL, Beyotime, Cat. No.C1006) for 5 min. Images were captured using a Zeiss Axio Observer fluorescence microscope (Zeiss, Germany), and 53BP1 foci were quantified using ImageJ software.

Phospho-kinase antibody array

The Human Phospho-Kinase Array Kit (Cat. No. ARY003B) from R&D Systems (Minneapolis, Minnesota, USA) was utilized to identify potential kinase activities. This array includes 43 kinase phosphorylation sites and two related total proteins. Cell lysate samples were diluted according to the manufacturer’s instructions and incubated with the array membranes overnight at 4 °C. Following incubation, the membranes were washed to remove unbound proteins and then incubated with a cocktail of biotinylated detection antibodies for 2 h at room temperature. Streptavidin-horseradish peroxidase was subsequently added. The detection of kinase activity was performed using chemiluminescent detection reagents and imaged with an ImageQuant LAS 4000 analyzer (GE Healthcare, Pittsburgh, Pennsylvania, USA).

Xenograft assay

The Animal Protection and Use Committee of Tongji University approved all protocols for the animal experiments described in this study. Forty female NOD scid gamma (NSG) mice, aged 4 weeks, were used. These mice were subcutaneously injected in the right abdomen with SW1116 cells that had been engineered to express either control shRNA (shCtrl), shRNA targeting FOXC1 (shFOXC1), or control shRNA with U0126 treatment (shCtrl + U0126), provided by Changzhou Cavens Model Animal Co., Ltd. The U0126 was administered intraperitoneally at a dose of 10.5 mg/kg daily. Once tumors were established, the mice were divided into two dietary groups: one receiving a normal control diet and the other a serine/glycine-deprived diet (Ser(-) diet). Three days after dietary modification, treatments commenced. Mice received either no drug treatment or were treated with 5-FU (40 mg/kg) following a cyclic regimen of three consecutive daily injections and four days of recovery. After 4 weeks, mice were euthanized, and tumors were excised for measurement and weighing. A portion of each tumor was fixed in 10% paraformaldehyde, paraffin-embedded for immunohistochemical analysis, and the remainder was used for protein extraction and serine level assessment.

Statistical analysis

Differences between groups were calculated using Student’s t-test, one-way analysis of variance, Chi-squared test, or Fisher’s exact test. GraphPad Prism version 8.0 was used for all statistical analyses. Data are presented as mean ± SD. NS: not significant. Significance is defined as *P < 0.05, **P < 0.01, ***P < 0.001.