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Fig. 5 Zebrafish parkin is stress-inducible and stress-protective.
(A) Zebrafish parkin is transcriptionally up-regulated in response to mitochondrial stress. Pac2 cells (zebrafish fibroblasts) were incubated with rotenone (10 nM) for 1 h and harvested 6 h after treatment. Total cellular RNA was isolated and subjected to semi-quantitative real-time PCR using zebrafish parkin-specific primers. The amount of RNA of each sample was normalized with respect to the endogenous housekeeping control gene β-actin. Shown is the fold increase in the amount of zebrafish parkin-specific mRNA compared with the untreated control based on 5 independent experiments. (B) Zebrafish parkin protects cells from excitotoxin-induced cell death. SH-SY5Y cells were co-transfected with EYFP and either vector, FLAG-tagged human parkin (hu-parkin) or FLAG-tagged zebrafish parkin (zf-parkin). 24 h after transfection, the cells were incubated with kainate (1 mM) for 3 h, fixed, permeabilized, and analyzed by indirect immunofluorescence using an antibody against active caspase-3. Shown is the percentage of apoptotic cells among at least 300 transfected cells. The quantification is based on three independent experiments performed in duplicates. As a control for parkin expression, aliquots of the cell lysates were immunoblotted with an anti-FLAG monoclonal antibody (lower panel). ***p<0.001