Fig. 6

Ethanol boosts the expression of GFAP and NF-κB p65 by a mechanism involving the activation of hemichannels and pannexons in cultured astrocytes. (A-I) Representative fluorescence micrographs of GFAP (white), NF-κB p65 (red) and DAPI (blue) labeling by astrocytes under control conditions (A-C) or treated for 24 h with 100 mM ethanol alone (D-F) or in combination with 50 µM gap19 (G-I). Insets: 2X magnification of the indicated area of panels C, F and I. (J) Quantitation of NF-κB p65 nuclear staining by astrocytes under control conditions (white bars) or treated for 24 h with 100 mM ethanol alone (red bars) or in combination with 50 µM gap19, 50 µM ¹⁰panx1 or 50 µM gap19 plus 50 µM ¹⁰panx1. (K) Quantification of Manders’ overlap coefficient for NF-κB p65 with DAPI by astrocytes under control conditions (white bars) or treated for 24 h with 100 mM ethanol alone (red bars) or in combination with 50 µM gap19, 50 µM ¹⁰panx1 or 50 µM gap19 plus 50 µM ¹⁰panx1. (L) Quantitation of GFAP staining normalized to control conditions (dashed line) by astrocytes treated for 24 h with 100 mM ethanol alone (red bars) or in combination with 50 µM gap19, 50 µM ¹⁰panx1 or 50 µM gap19 plus 50 µM ¹⁰panx1. ***p < 0.0005, ethanol treatment compared to control conditions, ^#p < 0.05, ^##p < 0.001, effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). Data were obtained from at least three independent experiments with three or more repeats each one (≥ 20 cells analyzed for each repeat). Calibration bar = 80 μm