2.1. Materials
C2-Cer, LPS, adenosine 5′-triphosphate (ATP) disodium salt hydrate, SN50, verapamil, imipramine, sulfo-N-succinimidyl oleate (SSO) and phorbol 12-myristate 13-acetate (PMA) were acquired from Sigma-Aldrich (St Louis, MO, USA). Anti-NLRP3, anti-TXNIP, anti-CD36, anti‑nuclear factor-κB (NF-κB) and anti-p-NF-κB antibodies were purchased from Abcam (San Francisco, CA, USA). The secondary antibody was obtained from ZSGB-BIO (Beijing, China). Anti-caspase-1, anti-β-actin, and lysis buffer were obtained from Cell Signaling Technology (Beverly, MA, USA).
2.2. Cell culture and treatment
Murine macrophage cell line J774A.1 and human monocyte leukemia cell line (THP-1) were obtained from the University of Science and Technology of China. In the subsequent experiments, THP-1 monocytes were stimulated with PMA (100 nM) for 24 h to induce them to differentiate into adherent macrophages. The J774A.1 cells and THP-1 macrophages were randomly divided into 1) normal control group, LPS/ATP group, imipramine intervention + LPS/ATP group (cells were incubated with 1 μg/mL LPS for 4 h, treated with 10 μmol/L imipramine for 3 h, and then with 5 mM ATP for 30 min), and imipramine control group (imipramine group); 2) normal control group, LPS/ATP group, verapamil intervention + LPS/ATP group (cells were incubated with 1 μg/mL LPS for 4 h, treated with 10 μmol/L verapamil for 3 h, and then with 5 mM ATP for 30 min), and verapamil control group (verapamil group); 3) normal control group, Cer group (30 μmol/L C2-Cer), verapamil intervention + Cer group (cells were pretreated with 10 μmol/L verapamil for 3 h and co-incubated with 30 μmol/L C2-Cer 5 h, and TXNIP siRNA + Cer group (cells were transfected with TXNIP siRNA and incubated with 30 μmol/L C2-Cer for 5 h; and 4) normal control group, Cer group (30 μmol/L C2-Cer), SSO intervention + Cer group (cells were pretreated with 200 μmol/L SSO for 1 h and co-incubated with 30 μmol/L C2-Cer for 5 h), SSO control group, SN50 (NF-κB inhibitor) intervention + Cer group (cells were pretreated with 36 μmol/L SN50 for 1 h and co-incubated with 30 μmol/L C2-Cer for 5 h), and SN50 control group.
2.3. Transfection
Control siRNA and TXNIP siRNA were synthesized by GenePharma (Shanghai, China). Cell transfection was performed using Lipofectamine® 3000 RNAiMax reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. The cells were collected for Western blotting and RT-PCR at 48 h post-transfection.
2.4. Cell viability assays
The cells were treated with imipramine (0, 25, 50, 75, 100 μmol/L), C2-Cer (0, 15, 30, 45, 60 μmol/L) and verapamil (0, 25, 50, 75, 100 μmol/L) for 24 h and then incubated with MTT (Beyotime, Jiangsu, China) for 4 h. Subsequently, dimethylsulfoxide (DMSO) (Beyotime, Jiangsu, China) was added, and the absorbance was measured by the microplate reader (BioTek, Winooski, VT, USA) at 490 nm.
2.5. Western blotting
An equal amount of protein (10-20 μg) was taken from each sample, loaded into the individual lane, and subjected to vertical SDS-PAGE electrophoresis (concentration gel voltage 50 V, 1 h, and separation gel voltage 100V, 1.5 h). The separated proteins were electrophoretically transferred at 200 mA for 3.5 h onto a PVDF membrane (Millipore Corporation, Billerica, MA, USA). The membrane was incubated with primary antibodies and corresponding secondary antibodies. Protein bands were scanned using the chemiluminescence imaging system (GE Healthcare, Bucks, UK).
2.6. Real-time PCR
Total RNA was extracted from cells using the Promega reagent (Promega, Beijing, China) according to the manufacturer’s instructions. Promega-A3500 kit was used for reverse transcription of RNA and quantitation of cDNA. The 2-ΔΔCt method was used to calculate the relative expression level of target genes normalized to GAPDH expression.
![](https://myfiles.space/user_files/58653_1b1c6aeb34a62c68/58653_custom_files/img1612888818.jpg)
2.7. ELISA
At the end of each treatment described under section 2.2, the culture medium was collected, and the concentrations of IL-1β and IL-18 were measured using ELISA kits (MultiSciences Biotechnology, Hangzhou, China); manufacturer’s instructions were followed. The absorbance of each well was measured on a microplate reader (BioTek) using the wavelength of 450 nm. Each determination was performed in triplicate, the values were averaged, and the cytokine content was calculated using standard curves.
2.8. Cer detection by immunofluorescence
At the end of the treatment (see section 2.2), the cells were fixed, permeabilized, and incubated with the anti-Cer antibody (1:500, ENZO, Switzerland) at 4°C for 12 h. Subsequently, the cells were incubated with the secondary antibody in the dark at room temperature for 1 h. After washing, cells were stained with DAPI in the dark for 10 min. Nikon Eclipse 90i Fluorescence Microscope system (Nikon, Japan) was used to visualize the cells and record the images. The quantification of Cer in THP-1 macrophages utilized the diacylglycerol (DAG) kinase assay, as mentioned earlier [19].
2.9. ASM activity measurement
The cells were lysed using a 1X mammalian lysis buffer and the samples were treated with ASM assay reagents (ASM assay kit, Abcam, San Francisco, CA, USA) according to the manufacturer’s instructions. After incubating for one hour at room temperature, fluorescence was detected using a microplate reader (BioTek) at the excitation and emission wavelength of 540 and 590nm, respectively. The fluorescence in the blank wells was used as a negative control.
2.10. NF-κB-p65 DNA-binding activity
The activation of the NF-κB complex was determined using an ELISA-based p65 transcription factor assay (Cayman Chemicals, Ann Arbor, MI, USA). The nuclear extract was fractionated using the extraction reagent kit (Thermo Fisher Scientific Inc., Rockford, USA) according to the manufacturer’s protocol. Oligonucleotides containing p65 uniform binding sites were immobilized in the wells and incubated with nuclear extracts. Finally, after incubation with specific primary and horseradish peroxidase (HRP)-conjugated secondary antibodies, the absorbance was measured at 450 nm.
2.11. Statistical analysis
All experimental data are presented as the mean ± SD, and statistical analysis was conducted using SPSS version 23.0. The statistical significance of differences between the groups was determined using a one-way analysis of variance (ANOVA). P<0.05 was considered to indicated statistical significance.