Srptektifii

To block the C×43 function, GFBLs were seeded on 6-well plates (42,000 cells/cm²) in their normal growth medium. At day 2 when cultures became confluent, culture medium was replaced with serum-free medium. At day 3, cells were treated with 150 μM of Gap27 (SRPTEKTIFII; Biomatik, Cambridge, ON, Canada) [3 (link),48 (link),49 (link)], equal molar amount of Gap26 (VCYDKSFPISHVR) [3 (link),48 (link)–50 (link)], or corresponding scrambled Gap27 (TFEPDRISITK) [33 (link)] or Gap26 (YSIVCKPHVFDRS) [50 (link)] control peptides, respectively, for up to 24 h before total RNA isolation or collection of cell lysates/conditioned medium for Western blotting. In a set of experiments, cells were treated with increasing concentrations (25, 50, 75, 100 μM) of meclofenamic acid (MFA; M4531, Sigma-Aldrich, St. Louis, MO, USA), a pharmacological connexin inhibitor [51 (link)], or corresponding amount of MFA diluent (dH₂O) for 24 h before RNA isolation.

To study Cx43 function, fibroblasts were seeded on 6-well plates in their normal growth medium as above. After 48 h, cells were serum-starved for 24 h, and then treated with Cx43 mimetic peptide Gap27 (150 μM; SRPTEKTIFII; Biomatik, Cambridge, ON, Canada) that corresponds to the second extracellular (E2) loop domain of Cx43, and blocks its GJ and HC functions^{38 (link),51 (link),52 (link)}, and Gap19 (250 and 400 μM; KQIEIKKFK; LifeTein, Hillsborough, NJ, USA) or TAT-Gap19 peptide (200, 400, 500, and 600 μM; YGRKKRRQRRR-KQIEIKKFK; LifeTein) that interacts with nine amino acids in the LT-domain of the cytoplasmic loop of Cx43 and specifically blocks its HC function without affecting GJs^{53 ,54 (link)}. Control samples were treated with scrambled control Gap27 peptide (TFEPDRISITK; Biomatik)^{80 (link)}, or mutated, function-deficient control TAT-Gap19 peptide (YGRKKRRQRRR-KQAEIKKFK; LeifTein)^{54 (link)}, respectively.