Fig 2: Cx26 protein localization in human hepatoma HepaRG cells exposed to GTX, NGTX, and NC chemicals. Human hepatoma HepaRG cells (n = 1 and N = 1; at least three images per well) were exposed to GTX, NGTX, and NC chemicals for 72 h. The area and intensity of the fluorescent signal was quantified via ImageJ software (version 1.52p, Bethesda, MD, USA) and normalized to the fluorescent signal of the respective solvent control (DMSO CTL or PBS CTL). Significant difference compared to the solvent control was calculated with a parametric one-way ANOVA followed by a Dunnett’s post-hoc test to correct for multiple comparisons. Data are expressed as mean ± standard deviation with * p = 0.05. Only images of significantly affected chemicals and 2NF are shown. Scale bar = 100 µM, 20× objective.

Fig 3: Time course of connexin immunoreactivity in the sensory epithelium of Cx26Sox10Cre mice.Maximal projection rendering of two consecutive midmodiolar confocal optical sections taken at 1 µm intervals in the basal cochlear turn of Cx26Sox10Cre mice (panels a–h) and control Cx26loxP/loxP mice (panels i–p) at P6, P9, P14 and P30. Expression of Cx26 (panels a–d, i–l) and Cx30 (panels e–h, m–p) was detected with selective antibodies (green) nuclei were stained with DAPI (blue) and actin filaments with Texas red conjugated phalloidin (red). TCC, tall columnar cells forming a transient structure, also known as Kölliker's organ; CC, cuboidal cells that replace TCC during the first two postnatal weeks; DC, Deiters' cells (also known as outer phalangeal cells); IHC, inner hair cell; IS, inner sulcus; SLi, spiral limbus; OHC, outer hair cells; OS, outer sulcus; PC, pillar cells forming the tunnel of Corti (TC); NS, Nuel's space; V, vas spiralis; BM, basilar membrane; SL, spiral ligament; scale bar, 50 µm.

Fig 4: Cx26, Cx32, and Cx43 protein expression in human hepatoma HepaRG cells compared to primary human hepatocytes (PHH). Proteins were extracted from human hepatoma HepaRG cells (n = 3 and N = 3) and PHH (n = 1 and N = 3) and immunoblotting was performed. Densiometric quantitative data were obtained via Image Lab 6.0.1 software (Bio-Rad, Hercules, CA, USA). Data were normalized to the total protein loading (Figure S1) and expressed as a ratio to PHH (if the target was expressed in PHH). Significant differences compared to PHH were calculated with a parametric one-way analysis of variance (ANOVA) followed by a Dunnett’s post-hoc test to correct for multiple comparisons. Data are expressed as mean ± standard deviation with * p = 0.05 and **** p = 0.0001. (1 = HepaRG batch 284; 2 = HepaRG batch 297; 3 = HepaRG batch 301; 4 = PHH).

Fig 5: Connexin (Cx) hemichannels (HCs) expressed in melanoma cells mediate the propagation of calcium (Ca2+) waves induced by focal photodynamic therapy (fPDT) in vivo. (A) Pooled results of fPDT trials in GCaMP6s-expressing dorsal skinfold chamber (DSC) tumors in the following conditions: Ca2+-free extracellular medium (CFEM) supplemented with ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 5 mM); normal extracellular medium containing 2 mM of Ca2+ (NEM, control); CFEM supplemented with EGTA (5 mM) plus carbenoxolone (CBX, 100 µM) or flufenamic acid (FFA, 100 µM) or TAT-Gap19 (150 µM) or abEC1.1m (1 µM). The histogram shows the area under GCaMP6s ?F/F0 traces (A, inset) computed between the onset of fPDT (t = 10 s) and the end of the observation time window (t = 80 s) for each bystander cell order (abscissa); pooled data [mean ± standard error of the mean (s.e.m.)] for n = 6 experiments in at least 2 different tumors for each condition. a.u., arbitrary units; *, p < 0.05; ***, p < 0.001, the Kruskal-Wallis test (for post hoc pairwise comparisons, see Table S1). Data for EGTA and NEM conditions are also shown in Figure 1C; (B–D) In vivo 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI) uptake experiments: GCaMP6s-expressing DSC melanomas were incubated with DAPI (5 µM) dissolved in: CFEM supplemented with 5 mM of EGTA; NEM; CFEM supplemented with 5 mM of EGTA plus FFA (100 µM) or TAT-Gap19 (150 µM) or abEC1.1m (1 µM). Fluorescence images were acquired at 5 min intervals up to 30 min; (B) Representative images acquired before (t = 0 min) and after 30 min of DAPI incubation in EGTA conditions; scale bar: 20 µm; (C) Relative variation of nuclear DAPI fluorescence intensity [F(t)/F0] in tumor cells vs. time during dye uptake in EGTA conditions (mean ± s.e.m., n = 20 cells, 2 tumors). The red dashed line was computed by data fitting with the shown function f(t) (parameter values: a = -0.1754, b = 0.0647 min-1); (D) Box plots showing the distributions of ?F = F(30 min) - F0 for DAPI measured in n = 12 nuclei for each condition. Red horizontal bars indicate the median. ***, p < 0.001, the Kruskal-Wallis test (for post hoc pairwise comparisons, see Table S2); (E) Representative western blots for Cx43 (top) and Cx26 (bottom) expression in tumors (T) derived from B16-F10 or B16-F10-GCaMP6s cells and grown in DSCs (denoted as B16-F10 T and B16-F10-GCaMP6s T, respectively) compared with B16-F10 or B16-F10-GCaMP6s cells grown in culture dishes; graphs on the right show the corresponding relative optical density (mean ± s.e.m., n = 4 independent experiments; **, p < 0.05, ANOVA on Ranks; ***, p < 0.001, ANOVA). Detailed information about the Western blotting can be found at Figure S9. (F) Confocal fluorescence images obtained by immunostaining with antibodies selective for Cx43 (top left, green), Cx26 (bottom left, green) and MelanA (right, red) in representative sections of melanomas grown in DSCs; nuclei were stained with DAPI; scale bar: 10 µm.