The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University.
The human umbilical vein endothelial cells (HUVECs) and U937 monocytes used in this study were both purchased from American Type Culture Collection (Manassas, VA, USA). HUVECs were cultured in human endothelial serum-free medium (Invitrogen, Carlsbad, CA, USA) with 20% fetal bovine serum (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen), 100 μg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), and 150 μg/ml endothelial cell growth supplement (Becton, Dickinson and Company, Frankin Lakes, NJ, USA). U937 monocytes were cultured in RPMI1640 medium (Invitrogen), which contains 20% fetal bovine serum (Invitrogen) and 100 U/ml penicillin-streptomycin (Invitrogen). HUVECs and U937 monocytes were both cultured in an incubator (5% CO2, 37°C, and 90% humidity) (Thermo Fisher Scientific, Waltham, MA, USA).
Cell counting kit-8 assay
Cell vitality was detected using cell counting kit-8 kit assays (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer’s instructions.
According to the requirements of each experiment, HUVECs and U937 monocytes were pre-treated with different chemicals, including 18-α-GA (a connexin channel inhibitor; 50 μM, for 1 hour; Sigma-Aldrich) and Gap 27 (a connexin mimetic peptide that inhibits Cx43 channel function; 300 μM, for 1 hour; Sigma-Aldrich); α, β-methylene ADP (APCP; a CD73 inhibitor; 300 μM, for 1 hour; Sigma-Aldrich), exogenous ATP (200 μM, for 1 hour; Sigma-Aldrich), and ADO (100 μM, for 1 hour; Sigma-Aldrich); fentanyl (10 μg/ml, for 24 hours; Yichang Humanwell Pharmaceutical Co., LTD, Yichang, Hubei, China), sufentanil (25 ng/ml, for 24 hours; Yichang Humanwell Pharmaceutical Co., LTD), and remifentanil (50 ng/ml, for 24 hours; Yichang Humanwell Pharmaceutical Co., LTD).
U937-HUVECs adhesion was detected according to procedures described previous studies: U937 monocytes were first labelled with calcein-acetoxymethyl ester (5 μM, Invitrogen) for 30 min in the incubator. The labelled U937 monocytes were then washed twice and resuspended in a serum-free medium. The labelled U937 monocytes were counted and poured onto confluent HUVEC monolayers, which had been pre-treated for 12 hours with recombinant mouse tumour necrosis factor α (10 ng/mL; Peprotech, Rocky Hill, NJ, USA). The plates were incubated for 1 hour and then rinsed twice slightly with the serum-free medium. Adherent U937 monocytes remained on HUVECs and were counted with a fluorescence microscope (Olympus IX71, Tokyo, Japan). Eight different 200× visual fields in each well were selected for analysis .
ATP and ADO release detection
ATP release was detected with ATP bioluminescence assay kits (Sigma-Aldrich). The supernatants of HUVEC and U937 monocyte cultures were harvested on ice. One hundred microliters of supernatant were added to 100 μl of ATP assay mix solution in 96-well culture plates. The luminescence was read by a fluorospectrophotometer (Cary Eclipse, FL0811M005; bio/chemi-luminescence mode). The ADO content was detected using related ELISA kits (Xinyu Biotechnology, Shanghai, China), according to the manufacturer’s instructions .
Cx43 expression was detected with western blotting. Proteins samples were quantified with Pierce ™ BCA Protein assay kits (Thermo Fisher Scientific, Inc.). The protein sample (25 μg) was added into SDS-PAGE, and then transferred onto a polyvinylidene fluoride membrane. After blocking with 5% milk for 1 hour at room temperature, the membranes were incubated with Cx43 antibody overnight at 4℃ (anti-Cx43; 1:3000; Cat: SAB4501174, Sigma-Aldrich) and anti-β-tubulin for 1 hour (1:10000; Cat: T4026, Sigma-Aldrich). Protein band sizes were estimated with Alpha View software (version number: 2.2.14407, Protein Simple, Santa Clara, CA, USA). The original blots were showed in Supplementary Figure 1.
Parachute dye-coupling assay
Parachute dye-coupling assays were used to detect gap junction function in HUVECs. HUVECs were grown to confluence. Donor cells were first labelled with calcein-AM (5 μM) in the incubator for 30 minutes. According to a donor:receiver ratio of 1:150, donor cells were seeded onto receiver cells. After 4 hours, the results were observed with a fluorescence microscope (Olympus DP73, Tokyo, Japan). The average number of receiver cells around every donor cell was counted; this reflected the function of Cx43 channels .
Statistical analysis was performed using SPSS 15.0 software (SPSS, Inc., Chicago, IL, USA). Multiple comparisons among groups were performed using repeated-measures one-way analyses of variance, followed by Tukey post hoc comparisons.