Fig. 3.

Hemichannels in lens fiber cells are opened by H₂O₂ and this opening is inhibited by dominant-negative Cx50 mutants. Primary chick lens cell cultures were infected with high-titer RCAS(A) retroviral vehicle (V) or recombinant RCAS(A) retroviruses containing Cx50, or Cx50 mutants E48K, P88S or H156N. (A) Whole-cell lysate extracts were prepared and then immunoblotted with anti-Cx50, anti-FLAG-tag or β-actin antibody. (B) After primary cultures reached maturation at ∼10–11 days of cell culturing, they were treated with or without 0.3 mM H₂O₂ for 20 min, and then an EtBr dye uptake assay was performed. The EtBr uptake and corresponding phase-contrast images of lens fiber cells (lentoids bodies) were taken at the same magnification by fluorescence microscopy. The average pixel density of 30 lentoid bodies was measured using ImageJ software. The data are presented as the mean±s.e.m. (n=3). **P<0.01; ***P<0.001. Scale bar: 100 μm.