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. 2013 Mar 22;20(7):920–930. doi: 10.1038/cdd.2013.19

Figure 4.

Figure 4

The interaction between PINK1 and Bcl-xL protects against CCCP-induced apoptosis. (a and b) Apoptosis evaluation upon PINK1 silencing. Either shSCR or shPINK1 cells were treated with CCCP followed by cleaved PARP immunoblotting (a) or FACS analysis (b). Densitometric analysis on cleaved PARP revealed, at 24 h CCCP, a significant increase of cell death in shPINK1 cells compared with control (5.78±1.21 versus 3.34±0.92, P=0.03). The percentage of TUNEL-positive cells at 24 h was also significantly higher in shPINK1 compared with shSCR cells (28.47±0.66 versus 19.38±0.55, P=0.04).(c and d) Apoptosis rescue by PINK1. shPINK1 cells were transfected with PINK1 wt or KDD or vector alone (pcDNA). After 24 h CCCP, cleaved PARP levels were significantly lower in presence of PINK1 wt compared with both empty vector (3.86±0.08 versus 5.34±0.07, P=0.003), and KDD (5.37±0.45, P=0.042). The proportion of TUNEL-positive cells also significantly decreased in presence of PINK1 wt compared with both control (16.79±0.81 versus 25.40±0.68, P=0.009), and KDD (22.77±0.27, P=0.02). (e and f) Apoptosis evaluation in Bcl-xL-silenced cells after PINK1 overexpression. SH-SY5Y cells subjected to Bcl-xL knockdown were transfected with PINK1 wt or empty vector (pcDNA) and then treated with CCCP for 24 h. Upon mitochondrial depolarization, the increase of cleaved PARP levels in PINK1 overexpressing cells was similar to that observed in cells transfected with the empty vector (3.54±0.61 versus 3.37±0.57, P=0.85); the same result was obtained analyzing the percentage of TUNEL-positive cells by FACS (47.56±2.51 versus 49.28±2.47, P=0.82). (g and h) Apoptosis rescue by Bcl-xL S62E. shPINK1 cells were transfected with Bcl-xL S62E or S62A, or with the empty vector. At 24 h CCCP, cleaved PARP levels were significantly lower in presence of Bcl-xL S62E compared with both Bcl-xL S62A (2.76±0.55 versus 4.03±0.50, P=0.042) and empty vector (4.87±0.81, P=0.02). Similarly, the percentage of TUNEL-positive cells was significantly lower in shPINK1 cells transfected with Bcl-xL S62E compared with both Bcl-xL S62A (13.76±0.13 versus 18.41±0.35, P=0.003) and vector alone (24.48±0.73, P=0.0008). *P-values<0.05