Preliminary experiment
Cell proliferation assay
HUVECs (FuHeng Cell Center, Shanghai, China, FH0278) were incubated with ticagrelor (0 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) clopidogrel (0 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM), separately, for 12 h, 24 h or 48 h. Then cell viability was determined by CCK-8 (Biosharp, BS350B).
Formal experiment
Cell culture and treatment
HUVECs were cultured in complete growth medium that was F12K containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin Solution at 37℃ with 5% CO2.
HUVECs were treated with complete growth medium supplemented with DMSO (as control), ticagrelor, clopidogrel, DMSO plus LPS and CD14, ticagrelor plus LPS and CD14, or clopidogrel plus LPS and CD14, separately, for 16 h. The concentrations of these compounds are shown in the table 1.
Cell proliferation assay
Cells were seeded in 96 well culture plates (2000 cells/well). After the cells were incubated with the indicated compounds for 16 h. Finally, cell viability was tested with CCK8 reagent. We evaluated cell viability by measured the absorbance at 450 nm.
Western blot assay
Whole cell extracts were lysed in RIPA Lysis buffer (Beyotime, P0013B) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Then protein concentration of lysates was determined by BCA protein concentration determination kit (Beyotime, P0010). Cell lysates containing equal amount protein were resolved on a 10%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a PVDF membrane (Millipore, IPVH00010). After separate incubation with rabbit anti-p65 (CST, #8242), rabbit anti-p-p65 (CST, #3033), rabbit anti-MMP2 (proteintech, 10373-2-AP), rabbit anti-MMP9 (proteintech, 10375-2-AP), rabbit anti-E-cadherin (proteintech, 20874-1-AP), rabbit anti-IKBa (abcam, Ab32518), rabbit anti-ICAM-1 (proteintech, 10831-1-AP), rabbit anti-VCAM-1 (Affinity, DF6082), rabbit anti-E-selectin (proteintech, 20894-1-AP), rabbit anti-GAPDH, mouse anti-P-selectin (proteintech, 60322-1-Ig), mouse anti-MCP-1 (Affinity, BF0678), followed by horseradish peroxidase-conjugated secondary antibody, the membranes were visualized by ECL chemiluminescence.
RNA Extraction and quantitative polymerase chain reaction (qPCR)
Total RNA was extracted using TRIzol reagent (Ambion, 15596-026), and reverse transcription was accomplished with HiScript Reverse Transcriptase (VAZYME, R101-01/02). The reverse transcription products were amplified with SYBR Green Master Mix (VAZYME, Q111-02) according to the manufacturer’s instructions. The data were normalized according to the level of GAPDH expression in each individual sample. The qPCR primers are listed in Table 2.
Immunofluorescence assay
The cells were incubated with the indicated compounds and then fixed for 15 min in 4% paraformaldehyde in 1×phosphate-buffered saline (PBS) pH 7.4. The fixed cells were permeabilized for 20 min with 0.5% Triton X-100 in 1×PBS and then blocked in 1× PBS with 1% bovine serum albumin for 30 min. The cells were incubated with the appropriate primary rabbit anti-p65 (CST, 8242S) and then stained with Alexa Fluor Cy3-labeled goat anti-rabbit immunoglobulin G (BOSTER, BA1032) and DAPI (Beyotime, C1002), separately. The subcellular localization of p65 was visualized using inverted fluorescence microscope (magnification, ×400).
Apoptosis assay
After incubated with the indicated compounds for 16 h, the cells were harvested and stained with APC/7-AAD apoptosis kit (SUNGENE BIOTECH, AO2001-11A-H), and then were analyzed by flow cytometry.
Cell cycle assay
After incubated with the indicated compounds for 16 h, the cells were harvested and stained with cell cycle kit (KeyGEN BioTECH, KGA512), and then were analyzed by flow cytometry.
Cell migration assay
After treated with the indicated compounds for 16 h, HUVECs were resuspended in serum-free F12K (2.5×105 cells/mL), 200 μL was added to the upper chambers, and complete growth medium was added to the lower chamber. After 24 h incubation, cells which migrated to the lower face of the membrane were fixed with 70% ethanol solution and stained by 0.5% crystal violet. After washed by PBS for 3 times, the migrating cells were observed under a microscope and photographed (magnification, ×200).
Matrigel assay
The cells were incubated with the indicated compounds for 16 h, and then cultured for 6 h in 24-well plates coated with matrigel. The cells were imaged under an inverted microscope (Nikon, ECLIPSE Ts2) at 100 magnification and the network length and width was quantified.
Statistical analyses
All data were expressed as the mean ± S.D. The statistical significance of data was assessed by an unpaired two-tailed t-test. A value of p < 0.05 was used as the standard for statistical significance. All experiments were repeated 3 times or more.