Microarray data and differential expression analysis
The microarray gene expression dataset GSE134025 from the Gene Expression Omnibus (GEO) database is based on the GPL20115 platform[6]. Differential expression analysis was performed with R software (package: ‘Limma’). Differentially expressed genes (DEGs) were identified by the following criteria: (1) |log2 (fold change)| > 1 and (2) adjusted P value < 0.05. When DEGs were duplicated, the most significant genes were retained. The heatmap and volcano plot were generated with R software (packages: ‘pheatmap’, ‘ggplot2’, and ‘ggrepel’).
Human colon tissues
Paraffin-embedded colon specimens from 89 UC patients and 5 healthy individuals were obtained from the First Affiliated Hospital of Soochow University (Jiangsu, China). The clinical data of each patient are provided in Supplementary Table 1. The diagnosis of UC was confirmed histopathologically by H&E staining, and the Mayo score, Ulcerative Colitis Endoscopic Index of Severity (UCEIS), haemoglobin (Hb) level, C-reactive protein (CRP) level, and erythrocyte sedimentation rate (ESR) were available[14] (Supplementary Tables 1, 4-6). Ethical approval (reference number: 2021-325) was obtained from the Ethics Committee of the First Affiliated Hospital of Soochow University, and informed consent was obtained from all participants in this research.
Establishment of experimental colitis models
Six- to- eight-week-old male C57/BL/6 mice (21.45 ± 1.12 g) and BALB/c mice (21.46 ± 1.25 g) were obtained from the Laboratory Animal Center of Soochow University. Animals were raised in compliance with regulations, and experiments were approved by the Ethics Committee of Soochow University (reference number: SUDA20210918A01).
Dextran sulfate sodium (DSS, MeilunBio, Dalian, China)-induced acute colitis was established as previously reported[15]. Briefly, control mice were provided sterile ddH2O, and others were provided sterile ddH2O containing 3% DSS for 8 days. A total of 20 C57/BL/6 mice were randomly divided into 3 groups: (1) Control (n = 6), (2) DSS + phosphate-buffered saline (PBS) (n = 7), and (3) DSS + α-methyl-DL-tryptophan (α-MT) (n = 7). Mice in the DSS + PBS group were intraperitoneally injected with 100 μl of PBS, while mice in the DSS + α-MT group were intraperitoneally injected with 100 μl of α-MT (Sigma–Aldrich, Bellefonte, USA, 20 mg/ml, dissolved in sterile saline) once a day for 7 days (Fig. S3A). Weight loss, diarrhoea, and bleeding were monitored daily. The grade of DSS-induced intestinal inflammation was assessed using the disease activity index (DAI) as previously described (Supplementary Table 7)[16]. Eight days after induction of colitis, DSS-challenged mice were sacrificed.
To establish oxazolone (OXZ; Sigma–Aldrich)-induced colitis, BALB/c mice were randomly divided into 3 groups: (1) Control (n = 6), (2) OXZ + PBS (n = 8), and (3) OXZ + α-MT (n = 7). OXZ-induced acute colitis was induced according to previous protocols[15]. Briefly, cutaneous sensitization to OXZ was induced in mice by application of 200 μl of 3% OXZ (dissolved in ethanol) for 2 consecutive days. Five days later, 150 μl 1% OXZ (dissolved in 50% ethanol) was carefully delivered into the colonic lumen by insertion of a catheter to a distance of 4 cm from the anus. Then, the mice were maintained in a vertical position for 30 s. Control mice were intrarectally administered 50% ethanol. Mice in the OXZ + PBS group and DSS + α-MT group were intraperitoneally injected with PBS and α-MT, respectively, once a day for 4 days (Fig. S3B). Weight loss, diarrhoea, bleeding, and DAI were recorded. Mice were finally killed on Day 3 after colitis induction.
Cell culture and treatment
Caco-2 cells obtained from ATCC (Manassas, Virginia, USA) were cultured as an adherent epithelial monolayer in Dulbecco's modified Eagle’s medium (DMEM) containing 20% foetal bovine serum (FBS) and 1% penicillin–streptomycin–amphotericin solution in a 37 ℃ humidified incubator (Thermo Fisher Scientific, Waltham, USA) with 5% CO2.
To establish a cell model of ferroptosis, Caco-2 cells were treated with RSL3 (MeilunBio, 15 μM) with or without ferrostatin-1 (Fer-1; Macklin, 4 μM) for 24 h. To explore the effect of SLC6A14 on Caco-2 cell ferroptosis, cells were treated with α-MT (2.5 mM) for 48 h and then treated with RSL3 (15 μM) for 24 h.
Cell transfection
SLC6A14-, PAK6-, and C/EBPβ-specific small interfering RNAs (siRNAs; RiboBio, Guangzhou, China) were transfected into Caco-2 cells for 48-72 h using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.
RNA sequencing and bioinformatic analysis
Total RNA was isolated from RSL3-induced Caco-2 cells transfected with si-SLC6A14-NC, si-SLC6A14-1, or si-SLC6A14-2 for poly(A) enrichment. mRNA transcripts with poly(A) tails were fragmented, reverse-transcribed into double-stranded cDNA, and inserted into a single-stranded circular DNA backbone. The qualified DNAs were sequenced on the DNBSEQ platform (Beijing Genomics Institute, Shenzhen, China). mRNA expression was obtained after quality control, filtering, and read alignment.
DEGs were identified by the following criteria: (1) |log2 (fold change)| > 0.585 and (2) adjusted P value < 0.05. The heatmap and volcano plot were generated with R software (packages: ‘pheatmap’, ‘ggplot2’, and ‘ggrepel’). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed with R software (packages: ‘clusterProfiler’, ‘org.Hs.eg.db’, ‘pathview’, and ‘topGO’).
Haematoxylin–eosin staining and histological scoring
For histopathological analysis, haematoxylin and eosin (H&E) staining (Beyotime, Shanghai, China) was performed according to the manufacturer’s protocols. Briefly, colon tissues of mice and humans were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. The paraffin-embedded colon tissues were sliced into 5 µm sections and stained sequentially with haematoxylin to visualize nuclei and eosin to visualize the cytoplasm. H&E staining was evaluated with the tissue damage index (TDI) as previously described (Supplementary Table 8)[17].
Immunohistochemistry
Immunohistochemical (IHC) staining was conducted as previously mentioned with a 3,3’-diaminobenzidine (DAB) Detection Kit (Gene Tech, Shanghai, China). Briefly, paraffin-embedded sections of colon tissue were incubated with the indicated primary antibodies overnight at 4 ℃ and then with the corresponding HRP-conjugated goat anti-mouse/rabbit IgG secondary antibody for 1 h at 37 ℃. IHC reactions were visualized by DAB staining (brown) and haematoxylin counterstaining (purple). The IHC sections were scored blindly by two experienced pathologists and additionally analysed with ImageJ (IHC Profile) with the H-score semiquantitative scoring system (Supplementary Table 9)[18]. The antibodies used for IHC analysis are shown in Supplementary Table 11.
Cell viability assay
Cell viability was evaluated with a Cell Counting Kit-8 (CCK-8; NCM, Suzhou, China) according to the manufacturer’s instructions. Briefly, (3-5)×103 Caco-2 cells were seeded in a 96-well plate with 100 μl of DMEM containing 20% FBS and incubated for 48 h. After the indicated treatments, 10 μl of CCK-8 reagent was added to each well and incubated at 37 ℃ for 3 h. The absorbance at 450 nm (A450) was measured using a microplate photometer (Biotek, Vermont, USA).
Total iron content assay
The total iron content in Caco-2 cells and mouse colon tissue was measured with an Iron Content Assay Kit (Solarbio, Beijing, China) according to the manufacturer’s protocols. Briefly, Caco-2 cells and the medium were centrifuged at 3000 × g for 15 min at room temperature. The precipitates were resuspended in 170 μl of iron extraction reagent and homogenized by sonication (ice bath; 20% power; ultrasonication 3 s, interval 3 s, 10 repeats). For analysis of mouse colon tissue, 100 mg of colon tissue was homogenized. The supernatant of each sample was obtained by centrifugation at 4 ℃ and 4000 × g for 10 min. The protein concentration (Cpr) in the supernatant was measured for normalization with a BCA protein assay (Beyotime). Next, the abovementioned supernatant (144 μl; ddH2O for the blank wells and 0.125 μmol/ml standard solution for the standard wells) was mixed with iron assay reagents (reagent one: 72 μl; reagent two: 144 μl) and then heated at 100 ℃ for 1 h. The inorganic phase was obtained after mixing with trichloromethane and centrifugation at 10000 rpm for 10 min at room temperature. The absorbance at 520 nm (A520) was measured. Thus, . The relative iron content was determined by normalization to the controls.
Reduced glutathione assay
The content of reduced GSH in Caco-2 cells and mouse colon tissue was measured with a Reduced GSH Assay Kit (Njjcbio, Nanjing, China) in accordance with the instructions. Briefly, the cell and colon samples were homogenized in 60 μl of RIPA lysis buffer and centrifuged at 4 °C and 8000 × g for 10 min. The protein concentration (Cpr) in the supernatant was measured for normalization. Then, the supernatant (30 μl) was mixed with Reagent One Application Buffer (120 μl) and centrifuged at 4 ℃ and 4000 rpm for 10 min. The absorbance at 420 nm (A420) was measured, and the GSH concentration (CGSH) was calculated according to the standard concentration curve. Thus, . The relative GSH content was determined by normalization to the controls.
Malondialdehyde assay
The malondialdehyde (MDA) content in Caco-2 cells and mouse colon tissues was assessed with an MDA Assay Kit (Beyotime) based on the manufacturer’s instructions. In brief, cell and colon samples were homogenized in 140 μl of RIPA lysis buffer and centrifuged at 4 °C and 12000 × g for 10 min. The protein concentration (Cpr) in the supernatant was measured for normalization. The supernatant (120 μl) was mixed with 240 μl of MDA assay buffer, incubated at 100 ℃ for 1 h and then centrifuged at 25 ℃ and 1000 ×g for 10 min. The absorbance at 532 nm (A532) was measured. Then, the MDA concentration (μmol/mg prot) was calculated according to the standard concentration curve, and the relative MDA content was determined by normalization to the controls.
Immunofluorescence staining
A total of (3-5)×103 Caco-2 cells were seeded on coverslips in a glass bottom dish (φ = 20 mm, NEST, Shenzhen, China) in 1 ml of DMEM containing 20% FBS and incubated for 48 h. After the indicated treatments, the cells were fixed with 4% PFA for 15 min at room temperature and then permeabilized by using 0.2% Triton X-100. The cells were incubated with a rabbit polyclonal anti-human/mouse prostaglandin-endoperoxide synthase 2 (PTGS2) antibody (Proteintech, Wuhan, China) overnight at 4 ℃ after blocking with 2% bovine serum albumin (BSA) for 1 h. Then, the cells were incubated with the corresponding Cy3-labelled goat anti-rabbit secondary antibody for 1 h at 25 ℃ and finally mounted in the confocal glass bottom dish with antifade medium containing 4',6-diamidino-2-phenylindole (DAPI; Beyotime). The targeted cells were observed with a laser scanning confocal microscope (Zeiss, Oberkochen, Germany). Relative PTGS2 expression was calculated by dividing the number of PTGS2+ cells (with red staining in the cytoplasm) by the number of DAPI+ cells (with blue staining in the nucleus) followed by normalization to the controls.
C11 BODIPY581/591 fluorescence assay
A total of (3-5)×103 Caco-2 cells were seeded on coverslips in a glass bottom dish and incubated for 48 h. After the indicated treatments, the cells were incubated with C11 BODIPY581/591 dye (ABclonal, Wuhan, China, 10 μM) at 37 ℃ for 1 h. The targeted cells were observed with a laser scanning confocal microscope after excess dye was removed by wiping with PBS. Quantitative analysis of stained regions was performed with ImageJ (NIH, MD, USA). C11-BODIPY581/591 levels were calculated as the ratio of the area with lipid oxidation (green) to the area without lipid oxidation (red).
Promoter prediction analysis
The promoter sequences of PAK6 were obtained from the GRCh38/hg38 human assembly via the University of California Santa Cruz (UCSC) Genome Browser (https://genome.ucsc.edu/cgi-bin/hgGateway). Then, the potential transcription factors and their binding sites were predicted with the TRANSFAC, ChIP-Atlas, and CIS-BP databases. The intersection of these three datasets was visualized with an interactive Venn diagram viewer, jvenn[19]. The predicted binding sites were visualized with the WebLogo program (http://weblogo.berkeley.edu/logo.cgi).
Dual-luciferase reporter assay
The relative luciferase activity of the C/EBPβ promoter was determined with a Dual-Luciferase Reporter Assay System (Promega, Wisconsin, USA) following the instruction manuals. In brief, Caco-2 cells were cotransfected with the C/EBPβ luciferase reporter plasmid (pGM C/EBPβ-Lu; Genomeditech, Shanghai, China) and SLC6A14-specific siRNAs with Lipofectamine 2000. After 48 h, the cells were lysed with passive lysis buffer (PLB), and firefly and Renilla luciferase activities were measured with a luminometer (Biotek). The relative luciferase activity was calculated by dividing the firefly luciferase activity by the Renilla luciferase activity and was normalized to si-SLC6A14-NC.
Chromatin immunoprecipitation assay
The chromatin immunoprecipitation (ChIP) assay was performed using an EZ-ChIP Kit (Sigma–Aldrich) according to the manuals, as previously reported. Briefly, RSL3-induced Caco-2 cells were cross-linked with 37% formaldehyde for 10 min. The cross-linked chromatin was sheared with the appropriate sonication protocol (ice bath; 10% power; ultrasonication 5 s, interval 10 s, 16-18 repeats) into fragments between 200 bp and 1000 bp in length, which were immunoprecipitated with a rabbit polyclonal anti-human/mouse C/EBPβ antibody (Proteintech). Normal mouse IgG was used for the negative control, while an anti-RNA polymerase antibody was used for the positive control. After the complexes were subjected to reverse cross-linking, the purified short hairpin DNA was detected by qPCR and DNA agarose gel electrophoresis (AGE) using primers specific for the PAK6 promoter region designed with Primer Premier 6 (Premier, Canada). The primers are listed in Supplementary Table 10.
Protein extraction and Western blot analysis
Caco-2 cells and mouse colon tissues (100 mg) were lysed with RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail. The total protein concentration was measured with a BCA protein assay kit. Equal amounts of protein (30 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a 0.45 µm polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% BSA for 1.5 h and incubated with the indicated primary antibodies overnight at 4 ℃. The following day, the membrane was incubated with the corresponding HRP-conjugated secondary antibodies at room temperature for 1 h. Finally, the membrane was visualized with ECL reagents in a ChemiDocTM MP Imaging System (Bio–Rad, CA, USA). The antibodies used in the Western blot analysis are listed in Supplementary Table 11.
Total RNA isolation and qRT–PCR assays
Total RNA from Caco-2 cells or mouse colon tissue (100 mg) was extracted with TRIzol reagent (Vazyme, Nanjing, China) according to the manufacturer’s protocol. For analysis of individual genes, cDNA was synthesized from 1.0 μg of total RNA with RTIII Super Mix with dsDNase (Monad, Guangdong, China) in a 20 μl volume system with the following thermal cycling conditions: 37 °C for 2 min, 55 °C for 5 min, 50 °C for 15 min, and 85 °C for 5 min. qPCR was performed with a CFX96 TouchTM Real-Time PCR System (Bio–Rad) with ChemoHS qPCR Mix (Monad). The PCR conditions were as follows: 1 cycle at 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. GAPDH was used as the control. PCR was performed with three technical replicates. The primers used for qRT–PCR are listed in Supplementary Table 10.
Statistical analysis
All statistical data were analysed with GraphPad Prism 8 (La Jolla, CA, USA). The results with normal distribution are presented as the means ± standard error of means (SEMs) and the non-normal results are presented as medians ± interquartile ranges (IQRs). An unpaired t test was used to analyse normal data with equal standard deviation (SD), Welch’s t test was used to analyse normal data without equal SD, and Mann Whitney u test was used to analyse non-normal data in two groups. Analysis of variance (ANOVA) was performed to analyse normal data with equal SD, Welch’s ANOVA analysis was used to analyse normal data without equal SD, and Kruskal-Wallis test was used to analyse non-normal data in multiple groups. Pearson correlation analysis was performed to analyse correlations between numerical normal data, while Spearman correlation analysis was performed in non-normal data in two groups. R2 represents the coefficient of determination, and r represents the Pearson/ Spearman correlation coefficient. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). All experiments in this study were performed in triplicate.