Dataset analyses

The RNA expression data and clinical information were retrieved from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo) (GSE76297 [15] and GSE107943 [16]) and The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/tcga). The data acquired from the TCGA and GEO database were transferred to Transcripts Per Kilobase Million (TPM) for further analysis.

The single-cell sequencing data of 8 samples from 5 patients with intrahepatic cholangiocarcinoma was acquired from GEO database (GSE138709 [17]). Seurat R package was used to analyze raw data from GSE138709 in R with R studio [18]. Cells were excluded in accordance with following criteria. (1) more than 20% mitochondria-related genes. (2) more than 6,000 or less than 500 genes expressed. 32,626 cells were retained for further analysis after normalization. Harmony R package was applied for batch integration [19, 20]. Then cells were clustered into 15 cell populations by FindClusters function (dims = 1:18, resolution = 0.4). The tSNE and UMAP analysis were performed for reduction and visualization of gene expression. The signatures from previous publications and CellMarker [17, 21, 22] were used for annotation.

Tumor Immune Estimation Resource (TIMER2.0) (http://timer.cistrome.org/) [23] was used to analyze pan cancers expression of ACSL4. TIMER2.0 and CIBERSORT [24] were applied to search the relationship between ACSL4 transcriptional level and immune cell infiltration in CHOL.

Immunohistochemistry staining of ACSL4 was collected from The Human Protein Atlas (HPA) database (http://www.proteinatlas.org/) [25].

TISIDB (http://cis.hku.hk/TISIDB/) [26] was used to investigate the association of ACSL4 with immunostimulators, immunoinhibitors, chemokines (or receptors) in CHOL.

LinkedOmics (http://www.linkedomics.org/login.php) [27] was used to analyze co-expressed genes associated with transcription of the ACSL4 gene in CHOL.

Human tissue samples

The tissue was from 40 CHOL patients who underwent surgery from 2013 to 2015 at The First Affiliated Hospital of Nanjing Medicine University. The patients were followed up until death or October 25, 2019. Our research was approved by the Ethics Committee of The Affiliated Hospital of Nanjing Medical University. The patients informed consent was obtained according to regional regulations.

Real-time quantitative PCR (qPCR)

The total RNA of cells and tissues was extracted by RNA-Quick Purification Kit (Yishan, China). HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, China) was used to reverse transcribe extracted RNA into cDNA. Then, qPCR was conducted with AceQ qPCR SYBR Green Master Mix (Vazyme, China) on the 7900HT Fast Real-Time PCR System (Applied Biosystems, MA, USA). The relative expression of ACSL4 was normalized to GAPDH by the 2−ΔΔCt method. The primer sequences are listed as follows. ACSL4 forward: 5’-CATCCCTGGAGCAGATACTCT-3’, ACSL4 reverse: 5’-TCACTTAGGATTTCCCTGGTCC-3’; GAPDH forward: 5’-GGAGCGAGATCCCTC CAAAAT-3’, GAPDH reverse: 5’-GGCTGTTGTCATACTTCTCATGG-3’.

Western blotting

RIPA Lysis Buffer (Beyotime, China) with PMSF is used to lyse tissue samples. Western blot was performed according to the manufacturer’s protocol. NcmECL Ultra (NCM Biotech, China) was applied to detect the level of protein. The following antibodies were used: anti-ACSL4 (22401-1-AP, Proteintech, China) and anti-GAPDH (ab8245, Abcam, UK).

Cell culture and transfection

Human intrahepatic biliary epithelial cell line (HiBEC), human cholangiocarcinoma cell line RBE, HCCC9810, HuCCT1 and QBC939 were used in our study. All the cells were cultured in DMEM High Glucose (Biochannel, China) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin solution (Biochannel, China) at 37 °C with 5% CO2. Small interfering RNAs (siRNAs) were synthesized by GenePharma (Shanghai, China). Lipofectamine™ 2000 (11,668,019, Invitrogen, USA) was used for transfection following the manufacturer’s protocol. The following siRNA sequences were used: ACSL4 sense 5′-GAGGCUUCCUAUCUGAUUATT-3′ and anti-sense 5′-UAAUCAGAUAGGAAGCCUCTT-3′ [28].

CCK8 assay

CCK8 assay kit (Dojindo, Japan) was used to assess the proliferation of cells. HuCCT1 or RBE cells transfected with negative control siRNA (si-NC) or si-ACSL4 were seeded in 96-well plates at 1 × 103 cells/well the night before. On each day of the subsequent days, 100 µ medium containing 10 µ CCK8 reagent was added into each well. After 2 h incubation, the absorbance at 450 nm was measured by Multi-Mode Microplate Reader (Biotek, USA).

EdU assay

EdU cell proliferation kit (Beyotime, China) was used for EdU assay to assess cell proliferation. HuCCT1 or RBE cells transfected with si-NC or si-ACSL4 were planted in 24-well plates at 80% confluence and cultured for 24 h, followed by 2 h incubation with 10µM EdU medium. The cells were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton in PBS for 15 min. Sequentially, the cells were stained with Alexa Fluor 555 azide for 30 min and DAPI for 10 min in the dark. The cells were photographed under a fluorescence microscope (Leica, Germany).

Clone formation assay

HuCCT1 or RBE cells transfected with si-NC or si-ACSL4 were seeded in 6-well plates at 1 × 103/well and incubated for 10 days. The cells were fixed and then stained with Crystal Violet Staining Solution (Beyotime, China) for 30 min. The colonies were counted and photographed.

Transwell assay

Transwell assays were performed for migration. 5 × 104 HuCCT1 or RBE cells transfected with si-NC or si-ACSL4 were seeded into Transwell BD Matrigel (Corning, USA). Serum-free medium was added to the upper layer and medium containing 20% fetal bovine serum was added to the lower layer. After 2 days incubation, migrating cells were fixed with 4% paraformaldehyde and then stained with Crystal Violet Staining Solution (Beyotime, China). The images were shot under a light microscope (Olympus, Japan).

Wound healing assay

HuCCT1 or RBE cells transfected with si-NC or si-ACSL4 were seeded in 6-well plates at 5 × 105 cells/well. When the density of cells reached 90-100%, we used a 200 µl pipette tip to scratch a straight wound. The images were taken by light microscopy (Olympus, Japan).

ROS and iron assay

ROS level was measured by Reactive Oxygen Species Assay Kit (Biosharp, China). Intracellular ferrous iron (Fe2+) level was measured by FerroOrange assay kit (Dojindo, Japan). The fluorescence intensity was detected by Multi-Mode Microplate Reader (Biotek, USA).

Statistical analysis

Data were analyzed using R 4.2.1 and GraphPad Prism 8. T-test was used for differences between the two groups and χ2 test was used to analyze correlations between ACSL4 expression and clinicopathological variables. Kaplan-Meier methods and log-rank test were applied to assess survival outcomes. Associations were evaluated using Spearman’s correlation analysis. Differences were considered statistically significant when *P < 0.05, **P < 0.01, or ***P < 0.001.