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

(A) 斑马鱼parkin基因在线粒体应激响应中发生转录上调。将Pac2细胞（斑马鱼成纤维细胞）用鱼藤酮（rotenone，10 nM）孵育1小时，并于处理后6小时收获细胞。提取细胞总RNA，使用斑马鱼parkin特异性引物进行半定量实时PCR（semi-quantitative real-time PCR）检测。以内源持家基因β-肌动蛋白（β-actin）为参照，对各样品的RNA总量进行归一化校正。本结果基于5次独立实验，展示了相较于未处理对照组，斑马鱼parkin特异性mRNA的相对表达倍数。
(B) 斑马鱼parkin可保护细胞免受兴奋性毒素诱导的细胞死亡。将SH-SY5Y细胞与EYFP以及空载体、FLAG标签人源parkin（hu-parkin）或FLAG标签斑马鱼parkin（zf-parkin）共转染。转染24小时后，将细胞用红藻氨酸（kainate，1 mM）孵育3小时，随后固定、透化，使用抗活化半胱天冬酶-3（active caspase-3）抗体通过间接免疫荧光（indirect immunofluorescence）进行分析。本结果展示了至少300个转染细胞中的凋亡细胞占比，定量分析基于3次独立重复实验（每组设置2个复孔）。为验证parkin的表达情况，取部分细胞裂解液使用抗FLAG单克隆抗体（anti-FLAG monoclonal antibody）进行免疫印迹检测（结果见下方泳道）。***p<0.001