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. 2015 Mar 19;29(7):2912–2929. doi: 10.1096/fj.14-268276

Figure 7.

Figure 7.

Parkin overexpression and MitoT rescue defective mitophagy and protect human primary SAECs against CS-induced cellular senescence. A) Representative images of cells show mitochondrial morphology in human primary SAECs treated with or without CSE (0.2%) for 10 days with alternate day treatment. Cells were stained with MitoTracker Red for mitochondria. B) Average MitoTracker Green fluorescence (Fmitogreen) was measured by FACS in normal and COPD SAECs treated with or without CSE (0. 2%) for 10 days. A.U., arbitrary units. *P < 0.05 vs. Normal. C) Representative images show SA-β-gal activity in SAECs treated with or without CSE. Cells were treated for 10 days followed by staining with SA-β-gal. D) Average fluorescent intensity of C12FDG (FC12FDG) was measured by FACS in SAECs. Con, control. *P < 0.05 vs. Con-Normal; #P < 0.05 vs. Con-COPD. E) Representative images of normal and COPD cells that were transfected with YFP-Parkin and treated with CCCP (10 µM) for 2 hours. SAECs were stained with Tom 20 (red) and DAPI (blue). Slanting lines on images indicate the areas assessed for fluorescence intensity. F) Corresponding line scan of fluorescence intensity shows the colocalization of YFP-Parkin with mitochondria. *P < 0.05 vs. Normal. G) Representative images of SAECs from normal subjects stained with γ-H2AX (green); DAPI was used to label nucleus (blue). Vec, vector. H) Average MitoTracker Green fluorescence (Fmitogreen) was measured by FACS in SAECs treated with or without CSE (0. 2%) for 15 days. *P < 0.05 vs. control vector; #P < 0.05 vs. CSE plus vector. I) Average fluorescent intensity of C12FDG (FC12FDG) was measured by FACS in SAECs. *P < 0.05 vs. control vector; #P < 0.05 vs. CSE plus vector. Vec is a vector, MitoT represents cells treated with MitoT, and Parkin represents cells transfected with mCherry-Parkin, whereas MitoT + Parkin represents cells transfected with Parkin and treated with MitoT with or without CSE (0.2%) treatment for 15 days. Data are shown as the means ± sem (n = 3). Scale bars, 10 μm (A and E), 100 μm (C), and 20 μm (G).