Fig. 1. The presence of undocked Cx43 hemichannels in mouse granulosa cells demonstrated by Lucifer yellow (LY) dye uptake or ATP release triggered by divalent-cation-free (DCF) solution or mechanical stimulation. (A-C) Representative experiments show LY uptake by wild-type granulosa cells in regular extracellular solution (A), DCF solution (B) or DCF solution with 200 µM carbenoxolone (CBX; C). The left column shows blue nuclear staining with Hoechst 33342, the middle column shows intracellular green fluorescence resulting from the uptake of LY and the right column shows the overlay. Scale bar, 10 µm. (D) Quantitative data, expressed as the percentage of cells loaded with LY relative to the total number of cells in a field of view, counted after Hoechst staining (mean ± s.e.m. of four to six different coverslips under each condition) of wild-type (WT) or Cx43-deficient (Cx43-KO) cells. (E) The data are plotted as the percentage of cells loaded with LY relative to the total number of cells in the field of view (mean ± s.e.m. of three to four different coverslips under each condition) after mechanical stimulation (mech). See Results for the blockers used. (D,E) ***P<0.001 versus baseline (one-way ANOVA and Tukey's post-hoc test). (F) Representative experiments show LY (middle) and rhodamine dextran (bottom) uptake by wild-type granulosa cells after mechanical stimulation. Cell numbers were counted by nuclear staining with Hoechst 33342 (top). Scale bar, 50 µm. (G) Data represent ATP release by wild-type and Cx43-deficient (Cx43-KO) granulosa cells triggered by DCF solution with or without 200 µM CBX. The data were normalized to baseline value (wild-type cells in regular ECS, broken line). ***P<0.001 versus baseline (two-way ANOVA and Bonferroni post-hoc test).