Gap26

From Sigma-Aldrich (St. Louis, MO, USA): Adenosine 5′-triphosphate disodium salt hydrate (ATP, A1852), DMEM (D6429), Hanks′ Balanced Salt Solution (HBSS, H4641), HEPES sodium salt (H7006), Monensin (M5273), Nystatin (N6261), Amiloride (PHR1839), Xestospongin C (X2628), MRS-2179 (M3808), U73122 (U6756), Thapsigargin (T9033), Ryanodine (559276), Carbenoxolone (C4790), ¹⁰Panx1 (SML2152), Bafilomycin A1 (19-148), Apyrase (A6237), Gap26 (SML3074). From Molecular probes (Eugene, OR, USA): Hoechst 33,342 (H1399), Propidium iodide (P1304MP). From Evrogene (Moscow, Russia): MMLV reverse transcriptase (SK022S), SYBR Green I PCR Master Mix (PK147L); Triethylammonium salt (TNP-ATP, Ann Arbor, MI, USA, 20902). From Thermo Fisher Scientific (Waltham, MA, USA): Fura-2AM (Cat. #F1221), Fetal Bovine Serum (10099141). Selenium nanoparticles (kindly provided by Dr. S.V. Gudkov, Prokhorov General Physics Institute of the Russian Academy of Sciences, Moscow, Russia).

NRK-52E cells, a cell line of rat kidney tubular epithelial cell origin, were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM/F-12 supplemented with 10% fetal bovine serum. Cells were grown at 37 °C in an atmosphere of 5% CO₂ in air^{48 (link)}, and then pretreated with connexin channel inhibitors heptanol, 2 mM, for 1 h (Sigma-Aldrich, a Cx43 uncoupler)^{49 (link)} or Gap26, 300 μM, for 1 h (Sigma-Aldrich, a connexin mimetic peptide)^{50 (link)}; a Cx43 expression enhancer, retinoic acid (RA), 10 μM, for 24 h (Sigma-Aldrich)^{4 (link)}; NAC (Sigma-Aldrich, a kind of ROS scavenger), 10 mM, for 1 h before H/R or/and LPS exposure. “Parachute” dye-coupling assay was performed as described below. The solvents of heptanol and RA were DMSO.

BEAS-2B cells, a kind of lung epithelial cell line was obtained from American Type Culture Collection (Manassas, VA, USA). Cells were grown at 37 °C in an atmosphere of 5 % CO₂ in air and cultured in DMEM/F-12 supplemented with 10 % fetal bovine serum. BEAS-2B cells were pretreated with connexin channel inhibitors gap26, 300 μM, for 1 h (Sigma-Aldrich, a connexinmimetic peptide) and a Cx43 expression enhancer, retinoic acid (RA) 10 μM, for 24 h (Sigma-Aldrich) before LPS (Sigma-Aldrich, 5 μg/ml, 24 h) treatment. Corresponding solvents of gap26 and RA are DMSO [22 (link), 23 (link)].
Propofol was at the concentration of 15 μM for 1 h before being subjected to LPS exposure. This concentration at 15 μM was based on our investigation which showed propofol at 15 μM can profoundly decrease dye coupling and also based on our previous study. This concentration of propofol (15 μM) is in the range of target plasma concentration of propofol 2–4 μg/ml (i.e., 11–22 μM) as used clinically during major surgeries [24 (link), 25 (link)].