Fig. 2.

Activation of Cx43 HCs in lens epithelial HLE-B3 cells by H₂O₂and UVB radiation. (A) HLE-B3 cells were treated with 0.3 mM H₂O₂ or 63 mJ/cm² UVB radiation for different time periods and followed by a dye uptake assay with EtBr and FITC-Dextran. The data are presented as the mean ± SEM. (n = 3). ****, P < 0.0001 (One-way ANOVA). (B) HLE-B3 transfected with Silencer™ Negative Control or Cx43 siRNA. Membrane extracts were subjected to immunoblotted with anti-Cx43CT, β-actin and GAPDH antibodies. Lower panel shows the normalized ratios of band intensities of Cx43 and β‐actin (n = 3). ****, P < 0.0001 (One-way ANOVA). (C) HLE-B3 cells were transfected with Silencer™ Negative Control or Cx43 siRNA by Lipofectamine RNAiMAX and then treated with 0.3 mM H₂O₂ or 63 mJ/cm² UVB radiation and followed by a dye uptake assay with EtBr and FITC-Dextran. The data are presented as the mean ± SEM. (n = 3). ****, P < 0.0001 (Two-way ANOVA). (D and E) HLE-B3 cells were treated with 0.3 mM H₂O₂(D) for 1.5 h or 63 mJ/cm² UVB radiation (E) for 1 h Cx43E2 antibody was pre-incubated for 30 min before exposure of H₂O₂ or UVB radiation, and a dye uptake assay was performed with 0.1 mM EtBr and FITC-Dextran. The data are presented as the mean ± SEM. (n = 3). **, P < 0.01; ****, P < 0.0001 (One-way ANOVA). Scale bar: 50 μm. At least three microphotographs of fluorescence fields were taken under microscope (Keyence BZ-X710) with a 20X objective and TxRed and GFP filters. The average pixel density of 30 random cells was measured by using NIH ImageJ software.