2.1 Cell culture and reagents
THP-1 (human monocytic cells) cell line was obtained from Zhong Qiao Xin Zhou Biotechnology Company (shanghai, china). The THP-1 cells were grown in suspension in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 0.05 mM 2-mercaptoethanol (Sigma, Saint Louis, USA), and cultured in a 5% CO2 incubator with humidified atmosphere at 37℃. During the experiment, 100 ng/ml PMA (phorbol 12-myristate 13-acetate) induced THP-1 cells to differentiate from monocytes into macrophages. After attachment of the bottom surface, PMA-contained medium were removed and cells were washed with D-Hanks. Then, cells were starved with serum-free RPMI 1640 medium for 24 h. The macrophages were treated with drugs according to the needs of experiments.
Phorbol 12-myristate 13-acetate (PMA,) and lipopolysaccharide (LPS) were purchased from Sigma (Saint Louis, USA). Lipofectamine TM 2000 transfection reagent and RNA isolation kit (TRIzol) were purchased from Invitrogen (California, USA). Annexin V-Alexa Fluor 647/PI Apoptosis Assay Kit (FMSAV647-100, FcMACS, NanJing, China) was used to detect cell apoptosis. HiScript Ⅱ Q-RT SuperMix for qPCR and ChamQ SYBR qPCR Master Mix (without ROX) were purchased from Vazyme (Nanjing, China). Cell Counting Kit-8 (CCK-8) was obtained from Yeasen Biotech company (Shanghai, China). TLR4 inhibitor Resatorvid (Res), NF-κB inhibitor BAY11-7082 (BAY), ERK inhibitor U0126, P38 inhibitor SB203580 (SB), JNK inhibitor SP600125 (SP) were acquired from Selleck Chemicals Company (Houston, USA). Antibodies against NF-κB p65, p-NF-κB, p38 MAPK, p-p38 MAPK, ERK, p-ERK, JNK, p-JNK were purchased from Cell Signaling Technologies (MA, USA). Antibodies to Tubulin and GAPDH were received from Proteintech (Chicago, USA). anti-supervillin (SVIL) was purchased from Sigma(Saint Louis, USA).
2.2 Real-time PCR (RT-PCR)
Total RNA was extracted from treated THP-1-derived macrophages by using TRIzol reagent. For cDNA synthesis, HiScript II Q-RT superMix was applied to reverse transcription reaction. The cDNA was used for qRT-PCR with SYBR green under standard reaction condition by Bio-Rad CFX Connect System (Bio-Rad, Hercules, CA, USA). The primers for SVIL, IL-1β, IL-6, TNF-α, GAPDH were provided by GENERAY (Shanghai, China) and the sequence were shown in Table 1.
2.3 Protein isolation and western blots analysis
The proteins were obtained from treated cells, which were lysed by RIPA buffer, which contained PMSF (1%), protease (10%) inhibitor and phosphatase (10%) inhibitor (Roche). and the concentration of protein was assessed by BCA (Bicinchoninic Acid) method. 30 μg of protein lysate was added to each lane and separated by SDS-PAGE, then transferred electrically to polyvinylidene fluoride (PVDF) membranes (Millipore). Then the PVDF membranes were blocked with 5% non-fat dry milk or BSA in TBST solution for 90 minutes and incubated with primary antibodies overnight at 4℃. Membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies for 1h. After washed with TBST, membranes were added with SuperBright Subpico ECL substrate and visually observed by Chemiluminescence gel imaging system (SYNGENE). The Band densities were quantified by GELPRO4 software. The dilution ratio of primary antibody as follow: SVIL (1:500), GAPDH (1:5000), Tubulin (1:5000), p-ERK1/2 (1:1000), p-P38 (1:1000), p-JNK (1:1000), p-NF-κB (1:1000), ERK1/2 (1:1000), P38 (1:1000), JNK (1:1000), NF-κB (1:1000).
2.4 Lentiviral encapsulation and infection
The sequences of shRNAs targeting SVIL (shSVIL) and a scrambled shRNA (shNC) were designed and constructed by Gene Pharma (shanghai, China). ShRNAs (shNC or shSVIL) and Lentiviral plasmids were packaged in HEK293FT cells with Lipofectamine 2000 and then lentiviral particles containing shRNA (shNC or shSVIL) were produced.
THP-1 cells were infected with Lentiviruses containing shNC or shSVIL by polybrene (8 μg/ml), then the infected cells were selected by puromycin (1 μg/ml) for about 2 weeks. Cells expressing green fluorescence were also differentiated by PMA and were used in subsequent experiments.
2.5 Flow cytometry analysis
The treated THP-1 Cells were harvested and stained with the Annexin V-FITC and PI to detect cell apoptosis. Briefly, the harvested cells were resuspended in 500 μl binding buffer (1x) and incubation with 5 μl Annexin V-FITC and 10 μl PI for 15 min in the dark, then cell apoptosis was detected by flow cytometry (BD FACS Calibur, NY) according to the manufacturer’s instructions.
Cell cycle distribution changes were also measured by flow cytometry (BD FACS Calibur, NY). The cells were collected and fixed with 75% ethanol and stored at 4℃.Then, cell cycle was detected by flow cytometry according to the manufacturer’s instructions.
2.6 Cell proliferation assay
Cell proliferation was measured by CCK-8. The cells were seeded into 96-well plates at a density of 1x104 cells/well and incubated at 37℃. Then, cells were treated with/without indicated drugs and indicated times. Next, 10 μl CCK-8 solution was added into each well and incubated for 2.5 h. The cells viability was measured by microplate reader at the absorbance of 450nm.
2.7 Statistical analysis
The data was analyzed with GraphPad Prism 5.0 by two-tailed t-test (comparison of two groups) or one-way analysis followed by Turkey tests (comparison of multiple groups). *p < 0.05 was considered to be statistically significant. In our study, each experiment was repeated more than three times respectively.