Reagents. The reagents used were RSL3 (Cayman, 1219810-16-8), Ferostatin-1 (Caymen, 17729), SYTOX green (EZBioscience, A0012-R2), BODIPY 665/676 (Thermo Fisher, BD Biosciences), BODIPY 581/591 C11 (Thermo Fisher, Cat #D3861), recombinant human IL-1β (Thermo Fisher, 200-01B), recombinant human TNF-α (Thermo Fisher, 300-01A), recombinant human TGF-β1 (Med Chem Express, HY-P70543), ulixertinib (BVD-523) (Med Chem Express, 869886-67-9), GSK1838705A(Med Chem Express, 1116235-97-2), MHY1485 (Med Chem Express, CAS No. : 326914-06-1), Lipo8000 (Beyotime, C-0533), and recombination human CXCL14 (Abcam, ab50043).
Human samples. Patients with an IBD diagnosis who underwent enteroscopy were enrolled in this study. Colonic specimens were obtained from endoscopic biopsies immediately after removal from patients. Control specimens were collected from people without any clinical complaints who were enrolled and underwent a routine endoscopic checkup. Participants were recruited from The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. Patients provided written informed consent and consented to the use of their samples anonymously for research.
Mice. Mice used in the study were housed in specific pathogen-free conditions and allowed free access to standard food and tap water. All animal protocols were approved by the Institutional Animal Care and Use Committees of Southern Medical University and were carried out in accordance with the relevant guidelines. C57BL/6 mice and S100a4-cre mice on a BALB/C background were originally obtained from the Jackson Laboratory (Jackson Laboratory stock no. 012641).
DSS-induced colitis and Histology. Mice used in this study were six-eight week-old. Control mice were matched for age and gender in each experiment. Eight mice were included in each group. Chronic colitis was induced in mice by drinking water supplemented with 2.5% dextran sulfate sodium (DSS) (MW = 36,000–50,000, MP, Biomedicals, California, USA). DSS was dissolved in distilled water and administered for seven days, followed by 14 days of normal drinking water. This cycle was repeated three times. control mice received tap water without DSS.
AAV9 construction and tail vein injection. CXCL14 knockdown and control recombinant Adeno-associated virus vectors 9 (AAV9) were constructed (GENECHEM Biotech). The administration procedures were performed according to previous studies.24 Brieflfly, 5 × 1010 physical particles of AAV in 200 µl of PBS were injected into the tail veins of 4-week old BALB/c and
S100a4-cre mice. Colon were removed for CXCL14 evaluation with western blots and IF.
Cell culture. The human intestinal fibrobalst (HIF) cell line was purchased from ScienCell (#2920), and the human intestinal CCD18 fibroblast cell line (Pricella CL-0591) was obtained from from Pricella. Cells were frozen at low passage and used within 2–3 months of thawing. HIF cells were cultured in FM (GIBCO) supplemented with 2% FBS (Gemini Bio-Products). CCD18 cells were cultured in DMEM (GIBCO) supplemented with 10% FBS. Both cell lines were cultured under conditions recommended in the introduction.
Immunofluorescence. For immunofluorescence staining, tissue sections were fixed in 4% paraformaldehyde/PBS or methanol, followed by protein blocking (DAKO). For intracellular markers, sections were permeated with Perm buffer (Ebioscience) for 20 min at 18°C. Staining was performed overnight with the following primary antibodies at 4°C: anti-CXCL14 (#df12377, Affinity) and anti-SCD1 (sc-515844, Santa Cruz Biotechnology). This was followed by treatment with the secondary antibodies anti-rabbit Alexa594 and anti-rat Alexa488 (Invitrogen) and counterstaining with DAPI (Vektor). Images were acquired using a fluorescent microscope (Leica).
ELISA. The levels of CXCL14 in the ECM were measured using commercial ELISA kits (Cloud-Clone Corp, SEB607Hu), according to the manufacturer’s instructions. Protein concentrations were calculated and recorded.
RT-qPCR. Total RNA was prepared with the reagent (EZBioscience) in accordance with the manufacturer’s instructions and converted to cDNA using the Reverse Transcription Supermix (Bio-Rad). cDNA was amplified by PCR using the All-in-One™ qPCR Mix (GeneCopoeia™, FulenGen), according to the manufacturer’s instructions. The PCR program was as follows: 95°C for 30 s; 40 cycles (each cycle: 95°C for 15 s and 55°C for 40 s). All primers synthesized by Qingke Biotechnology Co. are listed in Table 1.
Table 1
GENE | F | R |
human SCD1 | CCGGACACGGTCACCCGTTG | CGCCTTGCACGCTAGCTGGT |
humanSREBF1 | GCTGCTGACCGACATCGAA | GGGTGGGTCAAATAGGCCAG |
humanHMGC1 | CATTAGACCGCTGCTATTCTGTC | TTCAGCAACATCCGAGCTAGA |
human MVD | GGACCGGATTTGGCTGAATG | CCCATCCCGTGAGTTCCTC |
human FASN | AAGGACCTGTCTAGGTTTGATGC | TGGCTTCATAGGTGACTTCCA |
human FDFT1 | CCACCCCGAAGAGTTCTACAA | TGCGACTGGTCTGATTGAGATA |
human ACLY | ATCGGTTCAAGTATGCTCGGG | GACCAAGTTTTCCACGACGTT |
humanLPGAT1 | GGTTTGCCTTCATGGTCGTCA | CCAGAACCGCTTACTGTCCA |
human G6PD | CGAGGCCGTCACCAAGAAC | GTAGTGGTCGATGCGGTAGA |
humanELOVL6 | GCACCCGAACTAGGAGATACA | CCCCGGCAACCATGTCTTT |
humanELOVL2 | CTGCTCTCAATATGGCTGGGT | TCCCCTGCGCTGGTAAGAT |
humanELOVL5 | AGTGGTGTATAACCTTGGACTCA | ACCAGAGGACACGGATAATCTTC |
humanDGAT2 | ATTGCTGGCTCATCGCTGT | GGGAAAGTAGTCTCGAAAGTAGC |
human ACSL4 | ACTGGCCGACCTAAGGGAG | GCCAAAGGCAAGTAGCCAATA |
human ACSL1 | CTTATGGGCTTCGGAGCTTTT | CAAGTAGTGCGGATCTTCGTG |
human ACSL3 | ATGGAAAACCAACCTCATAGCAA | GCCATCCCAGTTATACCAGCAA |
human FADS1 | GTTATCCAGCGAAAGAAGTGGG | CCAATAGTGGCACATAAGTGAGG |
human FADS2 | GACCACGGCAAGAACTCAAAG | GAGGGTAGGAATCCAGCCATT |
human COX2 | CCCTGTTTCCATCACTCCCTC | GCAGGTCTTTTGGGCATCC |
human FSP1 | GATGAGCAACTTGGACAGCAA | CTGGGCTGCTTATCTGGGAAG |
human GCH1 | ACGAGCTGAACCTCCCTAAC | GAACCAAGTGATGCTCACACA |
humanGAPDH | GGAGCGAGATCCCTCCAAAAT | GGCTGTTGTCATACTTCTCATGG |
humanCXCL14 | GCTACAGCGACGTGAAGAAG | CCTATTCTTCGTAGACCCTGC |
Measurement of Lipid Peroxidation. Lipid peroxidation was analyzed using flow cytometry. The cells were seeded at an appropriate density in a six-well plate and cultured overnight in DMEM. Cells were stained with 5 µM BODIPY C11 (Cat #D3861; Thermo Fisher) or BODIPY 665/667 for 30 min after the indicated treatment. Labeled cells were resuspended in DMEM supplemented with 5% FBS and subjected to flow cytometry analysis.
Western Blotting. Cell lysates were resolved on SDS/PAGE gels and transferred to a nitrocellulose membrane. The membranes were incubated with 5% skim milk for 1 h at room temperature and then treated overnight with primary antibodies diluted in blocking buffer at 4°C. The following primary antibodies were used: CXCL14 (#df12377, Affinity), Col1A2 (M6940, Immunity), Phospho-Akt Ser473 (4060, CST), Akt (2920, CST), ERK (9102S, CST), p-ERK (4370S, CST), p-MEK (S217/221) (9154P, CST), MEK (11049-1-AP, Proteintech), α-actin (Ray Antibody, RM2007), GAPDH (Proteintech, 60004-1-Ig), Total S6K (2708, CST), Phospho-p70 S6 Kinase T421/S424 (ap0502, ABclonal), SREBP1 (SC-13551, Santa Cruz), SCD1 (sc-515844, Santa), IGF-1R (Proteintech, 20254-1-AP), IgG (7074P2, CST), GPX4 (67763-1-Ig), p-IGF-1R (AP0368, ABclonal), α-SMA (67735-1-Ig, Proteintech), p-mTOR (Ser2481) (#2974, CST). After three washes, the membranes were treated with goat anti-mouse HRP-conjugated antibody or donkey anti-rabbit HRP-conjugated antibody (Invitrogen) at room temperature for 1 h and subjected to chemiluminescence using Clarity Western ECL Substrate (Bio-Rad). An Amersham Imager 600 (GE Healthcare Life Sciences) was used for the final detection.
Lentiviral Transduction. A lentiviral virus containing the CXCL14 transgene (GFP-positive) was obtained from Sangon Biotech (Shanghai, China). SCD1 knockdown in 2920/CCD18 cells was performed using SCD shRNA (h) lentiviral particles (sc-36464-v, Santa Cruz). Lentiviral viruses were used to infect cells, which were seeded at 3×105 cells per well in a 6-well plate 24 h before infection. Polybrene (8 µg/mL) was also added to enhance lentiviral transduction efficiency. The medium was then changed after 24 h of incubation. The cells were placed under selection with the appropriate antibiotic (puromycin). Cells were infected and selected for puromycin resistance for two weeks before the experiments were performed.
Plasmid Transfection. A CXCL14 overexpression plasmid was purchased from Sangon Biotech (Shanghai, China). Transfection was performed according to the manufacturer’s recommendations using Lipo8000 (Beyotime, C-0533; Beyotime). Overexpression efficiency was confirmed at the time of harvest by RT-qPCR and/or western blotting.
siRNA Knockdown Experiments. All reagents were obtained from Sangon Biotech (Shanghai, China) and Gene Pharma (Shanghai, China). Transfection was performed according to the manufacturer’s recommendations using Lipo8000 (Beyotime, C-0533; Beyotime). Knockdown efficiencies were confirmed at the time of harvest using RT-qPCR and/or western blotting. siRNA sequences are shown in Table 2.
Table 2
Gene (Human) | F | R |
siCXCL14-1 | GCGAGGAGAAGAUGGUUAUTT | AUAACCAUCUUCUCCUCGCTT |
siCXCL14-2 | GGGUCCAAAUGCAAGUGCUTT | AGCACUUGCAUUUGGACCCTT |
siIGF-1R-1 | UAGUUGUAGAAGAGUUUCCAG | GGAAACUCUUCUACAACUACG |
siIGF-1R-2 | AUAAUUUGGGAUUGAAAGCAA | GCUUUCAAUCCCAAAUUAUGU |
Statistical Analyses. Data, where applicable, are expressed as mean ± SD from at least three independent experiments. Statistical analyses were performed using GraphPad Prism software (version 6.01, GraphPad). Statistical significance was set at P < 0.05.