Fig 2: Cx32 protein expression in human hepatoma HepaRG cells exposed GTX, NGTX, and NC chemicals. Human hepatoma HepaRG cells (n = 3 and N = 1) were exposed to GTX, NGTX, and NC chemicals for 72 h. 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 S4) according to Bio-Rad’s instructions [40] and expressed as a ratio to their respective solvent control (DMSO CTL or PBS CTL). Significant difference compared to the solvent control (DMSO CTL or PBS CTL) 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 and **** p ≤ 0.0001. Only blots containing significantly affected expression patterns are shown.

Fig 3: Cx32 protein localization in human hepatoma HepaRG cells exposed GTX, NGTX, and NC chemicals. Human hepatoma HepaRG cells (n = 1 and N = 1; at least 3 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 and **** p = 0.0001. Only images of significantly affected chemicals are shown. Scale bar = 100 µM, 20× objective.

Fig 4: Lactating Panx1-/- mice have fewer Cx32 gap junctions in the mammary gland.(A) Immunofluorescent analysis of mammary gland cryosections during early lactation for Cx26, Cx30 and Cx32 (red) and cytokeratin14 (green) revealed no change in Cx26 and Cx30 gap junctions in knockout mice, while fewer Cx32 gap junctions were observed compared to control mice. Hoescht (blue) denotes nuclei. Scale bar = 50 um. (B) Values represent the mean number of connexin plaques (red) relative to the pixel area of the nuclei (blue), multiplied by a factor of 1x102, per 0.3mm2 ± S.E.M. N = 5.

Fig 5: Lactating mammary glands of Cx26 knockdown mice exhibited a cross-talk reduction in Cx30 at parturition.(A) Western blot and (B) immunofluorescent analysis of mammary glands from control and Cre-treated mice at parturition revealed a significant decrease in Cx30 expression and a reduction in Cx30 gap junction plaques, while no change was observed in either Cx43 or Cx32. Arrows denote connexin plaques. *p<0.05. Values are mean levels ±SEM. N=6. Hoechst staining denotes the nuclei. Scale Bars = 50 µm.