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. 2010 Nov 26;18(5):769–782. doi: 10.1038/cdd.2010.142

Figure 6.

Figure 6

Parkin protects cells from ER stress-induced cell death. (a) Increased expression of wild-type (wt) parkin protects cells from ER stress-induced cell death. SH-SY5Y cells were co-transfected with EYFP (as a control) and wt parkin or the pathogenic parkin mutants G430D or ΔUBL. Twenty four hours after transfection, cells were incubated with 10 μM thapsigargin (TG) or 5 μg/ml tunicamycin (TM) at 37 °C for 8 h, fixed, permeabilized, and then the activation of caspase-3 was analyzed by indirect immunofluorescence using an anti-active caspase-3 pAb. Shown is the percentage of apoptotic cells among transfected cells. Parkin expression levels were determined by immunoblotting using the anti-parkin PRK8 mAb. Loading was controlled by re-probing the blots for β-actin (lower panel). (b) Parkin-deficient cells are more vulnerable to ER stress-induced cell death. SH-SY5Y cells were transfected with parkin-specific or control siRNA duplexes and co-transfected with EYFP (as a control) or siRNA-resistant wt parkin (rescue parkin). Three days later, the cells were stressed with TG (10 μM) for 8 h fixed, permeabilized, and then the activation of caspase-3 was analyzed by indirect immunofluorescence as described in A. Parkin expression levels were determined by immunoblotting using the anti-parkin PRK8 mAb. Loading was controlled by re-probing the blots for β-actin (lower panel). (c) Mouse embryonic fibroblasts (MEFs) derived from parkin-knockout mice are more vulnerable to ER stress than wt MEFs. MEFs from wt or parkin-knockout (ko) mice were stressed with TG (10 μM) for 16 h and then cellular viability was determined by the MTT assay. Shown is the relative viability of ko MEFs in comparison with wt MEFs after TG treatment. Quantification is based on five independent experiments. (d) Skin fibroblasts of patients carrying pathogenic mutations in the parkin gene are more vulnerable to ER stress. Skin fibroblasts from patients and control indivduals were stressed with tunicamycin (TM, 10 μM) for 24 h, fixed, permeabilized, and then the activation of caspase-3 was analyzed by indirect immunofluorescence as described in (a). ***P< 0.001, **P<0.01, *P<0.05