Reagents and antibodies
Reagents
Recombinant Human GZMA(YA9870) was purchased from atagenix (Wuhan, China); RSL3 (HY-100218A), Rp-cAMPS (HY-100530D), Dipyridamole (HY-B0312) and FITC-Dextran(HY-128868A) were purchased from MedChemExpress (Shanghai, China); GPX4-shRNA plasmid, CREB1-shRNA plasmid, CDX2 promoter plasmid and GPX4 promoter plasmid were purchased from Youbio (Hunan, China); Lipofectamine 8000(C0533) was purchased from Beyotime(Shanghai, China); Human Granzyme A ELISA Kit(ab255728)was purchased from Abcam (Abcam, UK).
Antibodies: Antibodies targeting GPX4(67763-1-Ig, 1:400 for IF, 1:2000 for WB), SLC7A11(26864-1-AP, 1:200 for IF, 1:2000 for WB), CDX2(60243-1-Ig, 1:400 for IF, 1:2000 for WB), CREB1(12208-1-AP, 1:200 for IF, 1:2000 for WB), PRKACA(27398-1-AP, 1:2000 for WB), ZO-1(21773-1-AP, 1:1000 for IF, 1:5000 for WB), Occludin(13409-1-AP, 1:1000 for IF, 1:2000 for WB), GAPDH(10494-1-AP, 1:5000 for WB) were from Proteintech Company (Wuhan, China); PRKACA(ER1706-65, 1:200 for IF) was purchased from HUABIO(Zhejiang, China); a-tublin(RM2007, 1:5000 for WB), b-actin(RM2001, 1:5000 for WB) were purchased from Ray antibody biotech(Beijing, China); Phospho-CREB1(Ser133) (YP0075, 1:2000 for WB), PDE4 (YP0668, 1:1000 for WB), Phospho-PDE4 (YP0668, 1:2000 for WB) were from Immunoway Research (Plano, USA); Phospho-PKA C (Thr197) (#4781, 1:1000 for WB) was from Cell Signaling Technology (Danvers, USA); Lamin A/C (sc-376248, 1:3000 for WB) was from Santa Cruz Biotechnology (Dallas, Texas, USA).
Cell culture and transfection
CaCO2, HT-29 and HEK293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% Fetal Bovine Serum (FBS), 100 mg/ml streptomycin and 100 U/ml penicillin to provide essential nutrients and maintain cell viability. Cells were placed in a carbon dioxide (CO2) incubator set to a standard culture condition of 37°C and 5% CO2. The plasmids were delivered into cells with lipofectamine 8000 according to manufacturer's instruction.
Enzyme-linked immunosorbent assay (Elisa)
Peripheral blood from IBD patients and healthy volunteers was collected by using a blood vessel containing coagulant. The serum was obtained by centrifugation at 3000 rpm for 5 min. Fresh colonic mucosa was weighed, clipped, and then lysate in PBS containing protease inhibitors with ultrasonic. After centrifugation at 12000 rpm for 15 min, the level of GZMA were determined by Human Granzyme A ELISA Kit (Abcam, ab255728) according to the manufacturer’s instruction.
RNA extraction and quantitative real-time PCR
Total RNA was isolated from CaCO2 and HT-29 cells using the EZ-press RNA Purification Kit (B0004D). Reverse transcripted into cDNA according to PrimeScript™ RT Master Mix (TAKARA, Japan). Quantitative PCR (qPCR) were carried out using the TB Green Premix Ex Taq ™ II (TAKARA, Japan) according to the manufacturer’s instructions. Primer used in this study was listed as followed: GPX4 forward,5’-GAGGCAAGACCGAAGTAAACTAC-3’; reverse, 5′-CCGAACTGGTTACACGGGAA-3′; SLC7A11 forward,5’-ACGGTGGTGTGTTTGCTGTCTC-3’; reverse,5’-GCTGGTAGAGGAGTGTGCTTGC-3’;CDX2 forward,5′-CTCGGCAGCCAAGTGAAAACCA-3′; reverse, 5′-GCTTTCCTCCGGATGGTGATGTA-3′; ZO-1 forward, 5’-TGTGAGTCCTTCAGCTGTGGAA-3’; reverse, 5′-GGAACTCAACACACCATTG-3; Occludin forward 5’-ACAAGCGGTTTTATCCAGAGTC-3’; reverse ,5′-GTCATCCACAGGCGAAGTTAAT-3;GAPDH, forward,5′-AACGGATTTGGTCGTATTGGG-3′, reverse, 5′-CCTGGAAGATGGTGATGGGAT-3′
Western blotting (WB) analysis
For protein extraction, CaCO2 and HT-29 cells were lysed using a RIPA lysis buffer (Biosharp, China), supplemented with protease inhibitors. The BCA method was used to determine the protein concentration. Nuclear and cytosolic proteins were extracted by nuclear extraction kit (Beyotime, China). To perform Western blot analysis, equal amounts of protein samples were loaded onto SDS-PAGE gels and subjected to electrophoresis. The separated proteins were then transferred onto a nitrocellulose membrane, which was blocked with 10% FBS (Solarbio, China) to prevent non-specific binding. Next, specific primary antibodies were applied against the target protein of interest. After incubation with secondary antibodies conjugated to an appropriate detection enzyme, the protein bands of interest were visualized using a chemiluminescent substrate (Perkin Elmer).
ROS detection
The intracellular ROS level was analyzed using the Reactive Oxygen Species Assay Kit (BL714A, Biosharp, Anhui, China) according to manufacturer's instruction.
In Vitro Permeability Assay.
As described previously[19], a total of 1 × 105 CaCO2 cells were reeded onto 0.4 µm porous Transwell polycarbonate membranes(Transwell 3401; Corning, NY, USA) pre-inserted in 12-well plates. Subsequently, the cells were cultured at 37°C in a humidified 5% CO2 atmosphere with the culture media refreshed every other day. When cells reached confluence (18–21 days after seeding), cells were exposed to GZMA (500nM) or RSL3(5um) for 48 hours. 1 mg/mL paracellular marker fluorescein isothiocyanate (FITC)-dextran (4 kDa) was added to the apical well of each insert and the monolayer was incubated for 2 hours at 37°C. The permeability of the monolayer to FITC-dextran was assessed by quantifying the fluorescence intensity in the lower chamber using an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The fluorescence values were converted into FITC-dextran concentrations (pg/mL) using a standard curve. A total of three independent experiments were conducted.
Luciferase reporter assay
HEK293T Cells were co-transfected with the Luc-GPX4 or Luc-CDX2 promoter luciferase reporter plasmid and internal control plasmid(pGL4.74) by using Lipofectamine 8000 according to manufacturer’s instruction. After 24 hours of transfection, GZMA (500nM) was added into cell to incubate for another 24 hours. The RLU were measured using Duo-Lite Luciferase Assay System (Vazyme, China).
Immunofluorescence (IF) analysis
HT-29 and CaCO2 cells were digested and reseeded at a density of 0.5*105/mL in 6-well plates overnight. After GZMA treatment, cells were fixed in 4% paraformaldehyde for 15 minutes, wash three times with PBS for 5 minutes and then blocked in 10% goat serum for 1 hour. Cells were incubated with the primary antibodies overnight at 4°C. For tissue slides, after deparaffinization, the blocking solution (PBS with 5% normal donkey or goat serum and 0.3% Triton X-100) was used to incubate for 30 minutes at room temperature. The further incubation was performed with the primary antibody overnight at 4°C. The next day, secondary antibody was used to incubate for 1 hour at room temperature after washing. The coverslips were mounted onto glass slides with prolong gold reagent after staining the nuclei with 4′,6-diamidino-2-phenylindole (DAPI). Stained cells were visualized using a laser scanning fluorescent microscope.
Immunoprecipitation
After treatment with GZMA for 1 hour, HT-29 or CaCO2 cells were lysed in RIPA for 15 mins and incubated with antibodies targeted PKA or IgG at 4°C overnight. The next day, Protein A/G beads was added into the lysate mixture to incubate another 2 hours. The lysate was centrifugated to remove the supernatant, the mixture was lysated with loading buffer and analyzed by Western blots.
Crypt isolation, Culture, and the Generation of Organoids
As described in our previous work[9], euthanizing the mice according to ethical regulations, the intestinal tissues were aseptically harvested. The surrounding mesentery and adipose tissues were removed by washing the intestines with colded DPBS. The intestinal tissues were then cut into 2 mm-wide sections and digested using Gentle Cell Dissociation Reagent cGMP (STEMCELL, 100–0485) on a shaker with a speed of 300rpm at 4°C to release crypts. The tissue suspension was filtered through a 70µm filter, and the collected tissue suspension passed through the filter and suspended with intestiCult™ Organoid Growth Medium (Mouse, STEMCELL, 06005) to culture. GZMA (500nM) and RSL3(5um) were added to observe the effect on intestinal organoids generation.
DSS-induced chronic colitis model
As described in our pervious study[20], mice aged 6–8 weeks were induced by 2% (w/v) dextran sulfate sodium (DSS, Millipore Corporation, Billerica, MA, USA) in drinking water for 7 days, and followed by GZMA or vehicle treatment. GZMA was administered via intraperitoneal injection at a dose of 20 µg per mouse on day 0, day 2, day 4, and day 6. The body weight changes, and overall survival were recorded daily. On day 7, mice were scarified, and colon was isolated for pathological analysis. All animal experiments were approved by Southern Medical University animal committee and performed at Southern Medical University.
Statistical analysis
Statistical analyses were performed with GraphPad Prism 6.0. Unless otherwise stated, data were presented as mean ± SD. P value < 0.05 was taken to indicate statistical significance.