Cell culture and X-ray radiation treatment

ESCC cell lines (TE1 and ECA109) were procured from the National Collection of Authenticated Cell Cultures and maintained in our laboratory. These cell lines were cultured in a controlled environment at 37 °C with 5% CO2. TE1 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS), while ECA109 cells were cultured in DMEM/high-glucose medium. ESCC cells were received 6MV X-ray radiation at a dose of 6–8 Gy, using a linear accelerator (Elekta Instruments, Inc., Stockholm, Sweden).

Lentiviral transfection for FGL1 expression modulation in ESCC cells

Lentiviral vectors were meticulously designed and constructed for both FGL1 overexpression (FGL1-OE) and knockdown (FGL1-KD) using a lentivirus system obtained from Hanbio Biotechnology Co., Ltd. (Shanghai, China). Plasmids carrying the target genes for FGL1 overexpression and the shRNA constructs for FGL1 knockdown were carefully prepared. Plasmids carrying the target genes for FGL1 overexpression and the shRNA constructs for FGL1 knockdown were carefully prepared. Plasmids carrying random nonsense sequences served as non-specific control (NC). These constructs were then transfected into HEK293T cells, chosen for their high transfection efficiency in lentivirus production. Over a 48-hour incubation period, HEK293T cells produced lentivirus particles containing the desired genetic alterations. The lentiviral supernatants were collected from the cell culture medium and subsequently used to infect ESCC cells. Stable ESCC cell lines with modified FGL1 expression were established through puromycin selection. The success of the transfection and its resulting impact on FGL1 expression were verified through molecular techniques, including PCR and western blotting analysis. This comprehensive approach ensured the successful establishment of an ESCC cellular model with controlled FGL1 expression levels, which serves as a crucial foundation for subsequent experimental investigations.

RNA isolation and quantitative real-time PCR (qRT-PCR)

The cDNA was synthesized from isolated RNA samples using the PrimeScript™ RT Reagent Kit (Takara Bio Inc., Shiga, Japan). qRT-PCR was performed using SYBR Premix Ex Taq™ II (Takara Bio Inc., Shiga, Japan), with β-actin used as the internal reference for normalization. Detailed primer sequences are provided in Table S1.

shRNA transfection

For IMPDH1 or Forkhead box O4 (FOXO4) knockdown, shRNAs targeting IMPDH1( sh-IMPDH1), FOXO4 (sh-FOXO4) and non-specific control (sh-NC) were sourced from Genechem Co., Ltd.(Shanghai, China). Transfection was conducted employing Lipofectamine™3000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) following established protocols. Briefly, selected shRNA sequences were complexed with Lipofectamine™3000 and added to the cells, facilitating shRNA uptake. Post-transfection for 72 h, the culture medium was refreshed to remove the transfection reagent. Detailed shRNA sequences are provided in Table S2.

Protein analysis by western blotting

Our western blotting experiments adhered to established protocols [31]. In brief, we extracted proteins from ESCC cells using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) and subsequently subjected them to electrophoresis on a 15% SDS-PAGE gel. These proteins were then transferred onto a PVDF membrane. Prior to antibody hybridization, the membranes were cut to focus on the target bands. Following blocking, we incubated the membranes with primary antibodies, including anti-FGL1 (1:1000, ab197357, Abcam, Cambridge, United Kingdom), anti-FOXO4 (1:1000, 21535-1-AP, Proteintech, Rosemont, IL, USA) and anti-IMPDH1 (1:1000, 22092-1-AP, Proteintech, Rosemont, IL, USA), for 14 h. Subsequently, we employed secondary antibodies for a 1-hour incubation at room temperature. Signal detection was conducted using the ECL kit (Millipore, Massachusetts, USA). Data analysis was performed using NIH-ImageJ software.

Site-directed mutagenesis and reporter gene assay

Site-directed mutations of the FOXO4- binding site within the FGL1 promoter region was performed by Hanbio Biotechnology (Hanbio Biotechnology Co., Ltd., Shanghai, China). The gene sequences of h-FGL1-pro-WT and h-FGL1-pro-MUT are in Appendix file 2. We constructed the firefly Luciferase (Fluc) reporter vector of h-FGL1-pro-WT and h-FGL1-pro-MUT as well as pcDNA3.1-FOXO4 overexpression vector. The constructed vectors were confirmed with DNA sequencing. Transcriptional activity was evaluated through the application of a luciferase assay system. The h-FGL1-pro-WT or MUT vector and pcDNA3.1-FOXO4 were co-transfected into 293T cells, with ranilla luciferase (Rluc) RL-TK used as an internal control. After a 48-hour incubation period following transfection, the quantification of fluorescence intensities was executed using the Dual-Luciferase® Reporter Assay System (Hanbio Biotechnology Co., Ltd., Shanghai, China).

Cellular immunofluorescence staining

ESCC cells were cultured in laser confocal-specific cell culture dishes (801,001, NEST Life Science Technology Co., Ltd., Wuxi, Jiangsu, China) until they adhered to the surface. Subsequently, they were washed with phosphate-buffered saline (PBS) and air-dried. Fixation was carried out using 4% paraformaldehyde for 20 min. Following fixation, permeabilization was achieved by employing a 0.1% Triton X-100 solution. Following a thorough washing step, the cells underwent a 30-minute blocking procedure at room temperature using a 5% bovine serum albumin solution. Subsequently, the cells were incubated with primary anti-FGL1 antibodies (1:1000, ab197357, Abcam, Cambridge, UK) at 4 °C overnight. Cells were incubated with secondary antibodies conjugated with fluorescent markers (1:2000, ab150077, Abcam, Cambridge, United Kingdom) at room temperature for 1 h. DAPI (C0065-10ML, Beijing Solarbio Science & Technology Co., Ltd, Beijing, China) was used to visualize the cell nuclei for 10 min. Visualization and imaging were performed using a laser confocal fluorescence microscopy (NIKON A1, Nikon, Tokyo, Japan).

Transwell invasion assay

A Transwell invasion assay was employed to evaluate cell invasiveness. Prior to cell inoculation, 50 µL of Matrigel (BD Biosciences, San Jose, California, USA) was applied to the upper membrane of the Transwell chamber. The cells designated for testing were prepared as a single-cell suspension without serum. A total of 2 × 105 cells were seeded into the upper compartment of the Transwell unit (8-micron pore size, Corning, New York, USA), and a medium containing 10% FBS was added to the lower chamber. The inoculated Transwell chambers were then incubated in a cell culture incubator. After 24 h, the Transwell chambers were carefully removed, and cells on the upper side of the membrane were gently swabbed. Cells that had migrated through the Transwell membrane were fixed with a 4% paraformaldehyde solution and stained with 0.05% crystal violet. Subsequently, they were observed under a microscope.

Wound healing assays

A wound healing assay was conducted to evaluate cell migration capabilities. The cells to be tested were cultured in a 6-well plate until they reached approximately 80% confluence. Once the cells had adhered to the surface and reached this confluence, a sterile pipette tip was used to create uniform scratches in the monolayer. The detached cells were removed using PBS, and serum-free medium was added to sustain further growth. The extent of cell migration into the scratched area was observed at specific time points, and the width of the scratch was measured.

RNA sequencing

Total RNA samples were extracted, subjected to agarose gel electrophoresis, and quantified using Nanodrop. mRNA enrichment was performed using oligo(dT) magnetic beads. RNA sequencing libraries were prepared using a kit, including RNA fragmentation, first-strand cDNA synthesis using random primers, second-strand cDNA synthesis with dUTP incorporation, end repair, A-tailing, and adapter ligation for Illumina sequencing. The final library was generated through PCR amplification. Quality control of the constructed libraries was conducted using the Agilent 2100 Bioanalyzer, followed by library quantification using qPCR methods. Sequencing of the prepared libraries was performed using the Illumina NovaSeq 6000 sequencer. The sequenced data were submitted to the GEO database for further analysis (GSE264221).

Animal lung metastasis assessment

Four-week-old male BALB/c nude mice were procured from the Animal Experiment Center at the School of Medicine, Xi’an Jiaotong University, China. The objective was to evaluate how the absence of FGL1 influences the metastatic potential of ESCC cells. To achieve this, the mice received tail vein injections of 5 × 107 ECA109 cells. The experimental group received FGL1-KD ECA109 cells, while the control group received NC ECA109 cells. After the manifestation of lung metastasis in the mice, humane euthanasia was performed, followed by surgical removal of lung tissues, and subsequent calculation of metastatic nodules. The euthanasia procedure adhered to ethical guidelines and utilized the carbon dioxide (CO2) inhalation method, as recommended by the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition. Animals were placed in a chamber pre-filled with CO2 gas at a concentration of 30% of the chamber volume per minute, ensuring rapid and painless euthanasia while minimizing animal suffering. All experimental protocols were conducted in strict accordance with the ethical guidelines and regulations approved by the Medical Ethics Committee of Xi’an Jiaotong University.

Statistical analysis

Statistical analysis was conducted utilizing GraphPad Prism 9.5.1 software. Data distribution normality was assessed using the Shapiro-Wilk test. Results are presented as mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical distinctions were determined using Student’s t-test or one-way analysis of variance (ANOVA) for data that adhered to normal distribution. In instances where the data deviated from a normal distribution, the non-parametric Mann-Whitney U test was employed, and outcomes were reported as medians. We employed Logrank tests to evaluate the significance of Kaplan-Meier survival curves. All statistical tests were two-tailed, and a p-value below 0.05 was deemed statistically significant.