Fig. 3

Involvement of TLR4 and TNF-α signaling in the LPS-induced enhancement of Cx26 hemichannel activity. a Real-time qRT-PCR experiments for TLR4 mRNA expression in Calu-3 cells and PBEPCs (n = 3 biological replicates or donors, respectively). b Immunofluorescence staining against TLR4 (yellow) in Calu-3 cells and PBEPCs. Scale bar = 20 μm. c EtdBr dye uptake rates in absence of [Ca²⁺]_ex relative to the rates obtained in presence of [Ca²⁺]_ex in Calu-3 cells treated for 24 h with vehicle, 1 ng/mL LPS ± preincubation (0.5 h) with 20 µM C34 or C34 alone (n = cell patches). Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.001 *** vs. vehicle, p < 0.001 ### vs. LPS). d Real-time qRT-PCR for TNF-α mRNA amount in Calu-3 cells treated for 3 h with 1 µg/mL LPS (n = 3). Unpaired two-tailed Student’s t-test. e EtdBr dye uptake rates in absence of [Ca²⁺]_ex relative to the rates obtained in presence of [Ca²⁺]_ex in Calu-3 cells treated for 24 h with vehicle, 1 ng/mL LPS ± preincubation (0.5 h) with 10 µM SPD-304 or 10 µM Marimastat, with SPD-304/Marimastat alone or for 1 h with 10 ng/mL TNF-α (n = cell patches). Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.05 *, p < 0.001 *** vs. vehicle; p < 0.001 ### vs. LPS; p < 0.001 +++ vs. TNF-α). f EtdBr dye uptake rates in absence of [Ca²⁺]_ex relative to the rates obtained in presence of [Ca²⁺]_ex in Calu-3 cells in which Cx26 expression was suppressed using respective siRNA and which were treated with 10 ng/mL TNF-α or vehicle for 1 h (n = cell patches). Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.01 **, p < 0.001 *** vs. negative siRNA + vehicle; p < 0.05 #, p < 0.001 ### vs. respective siRNA + vehicle; p < 0.001 +++ vs. TNF-α)