A Novel Role for Connexin Hemichannel in Oxidative Stress and Smoking-Induced Cell Injury
Figure 5
Hemichannel mediates cell death under oxidative stress as evidenced by gene transfection and silencing techniques.
(A). Cx43-GFP transfected N2A cells produced hemichannels and gap junctions (B, white arrow indicating typical distribution of Cx-channels in the cell-cell contact regions) and they responded to low Ca++ medium (E, LY uptake) and oxidative stress (C, EtBr uptake induced by CSE). The open hemichannels permit direct entry of ROS into the cells as detected by ROS sensitive dye (F). Live/dead assay showed significant cell death induced by CSE in Cx-transfected N2A cells (33%) (H,J) compared against their wild type N2A cells (9%) (G,I) (*p<0.0072). Scale bars (A–D): 5 µm, (E–J): 20 µm. (B). Apoptosis assay on Cx43 silenced MC after 10 hrs of induced oxidative stress (10% CSE). (A) Wild type MC in normal media (control) show live cells (blue nuclei, Hoechst 33342 stained). (B) Wild type MC in CSE show apoptotic cells (green cells, YO-PRO stained) indicated by white arrows. (C) Cx43 silenced MC show live cells (blue nuclei) and few necrotic cells (Red nuclei, Propidium Iodide) indicated by orange arrows. (D) Negative-siRNA transfected MC shows more apoptotic and necrotic cells than the wild type MC. Histogram shows the % apoptosis observed in different treatment groups (*p<0.0001). Scale bar: 5 µm