p66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke

Figure 3

p66Shc silencing improved CSE-induced mitochondrial dysfunction of airway epithelial cells. Beas-2b cells were transiently transfected with p66Shc siRNA for 24 h, and the silencing effect was confirmed by real-time PCR and Western blot (a). Transfected Beas-2b cells were further stimulated with 7.5% CSE for 24 h, and Western blot revealed that p66Shc siRNA significantly suppressed the increasement of p66Shc and p-p6Shc expression in the whole cell lysates (b and d) and notably reduced p66Shc mitochondrial translocation and the release of cytochrome c from the mitochondria (c and e). Quantitative analysis of protein expression as shown in (d) and (e) was carried out by Bandscan 5.0 software. Mitochondrial reactive oxygen species (ROS) was observed by confocal microscopy (f: upper row), and mitochondrial membrane potential (MMP) was determined by flow cytometry (f: lower row). Quantitative analysis showed that p66Shc silencing reduced mitochondrial ROS generation (g) and elevated MMP level (h) in Beas-2b cells exposed to 7.5% CSE. Firefly luciferase-based ATP assay revealed that p66Shc siRNA increased intracellular ATP levels in Beas-2b cells stimulated with 7.5% CSE for 24 h (i). All values are showed as the mean ± SD from three independent experiments. versus the control group and versus the 7.5% CSE-treated group.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)