As shown in Figure 1, mitochondrial length in SH-SY5Y cells remained almost the same after fixation for 30 min, and no excessive mitochondrial fragmentation was observed, suggesting that mitochondrial morphology in live cells is well reserved by fixation. Similar results were seen in other two cell lines. It is suggested that the mitochondrial fragmentation induced by a-synuclein overexpression may be due to its localization on mitochondria. To examine whether a-synuclein localization on mitochondria was promoted after SNCA transfection in our experiment, we isolated mitochondria and analyzed mitochondrial a-synuclein expression by immunoblotting. Mitochondrial or cytosolic extracts were first examined by immunoblotting for the presence of cytochrome oxidase subunit IV and b-actin. A robust band of COX IV was detected from the mitochondrial extracts, while the b-actin band was pretty weak; they were opposite in the cytosolic fraction, suggesting that the mitochondrial fraction was enriched with mitochondria, while few mitochondria were detected in the cytosolic fraction. a-Synuclein expression in mitochondrial pellets was very low in SH-SY5Y, PC12 and Hela cells, yet it was remarkably increased in all three cell lines after SNCA transfection for 48 h. Representative double-staining images of mitochondria and asynuclein demonstrated that after SNCA transfection for Nimorazole, asynuclein expression in all three cell lines was significantly increased and its distribution was greatly changed compared with those in the cells transfected with an empty vector. a-Synuclein was highly enriched in the nucleus in Hela and PC12 cells after SNCA transfection, and its expression was also strong in the nucleus in SH-SY5Y cells, but even stronger in the cytoplasm. a-Synuclein was expressed all over the cytoplasm in PC12 cells, where some peculiar punctate staining of a-synuclein was more standout and mostly co-localized with mitochondria, as shown in the amplified images in Figure S3. Cytofluorogram analysis of those double-staining images demonstrated that in all three cell lines, PC coefficients in a-synuclein overexpressed cells were magnificently elevated compared with the cells transfected with an empty vector. These indicate that more a-synuclein co-localizes with mitochondria in a-synuclein overexpressed cells. Although mitochondrial localization of a-synuclein was increased following SNCA transfection,Euphorbia factor L3 mitochondria in three cell lines still remain an elongated tubular and thread-like shape and no significant change in mitochondrial length was detected between a-synuclein overexpressed cells and those transfected with an empty vector. This indicates that a-synuclein overexpression increases its localization on mitochondria without affecting mitochondrial morphology in cell lines. Mitochondria undergo continuous fission and fusion in living cells, which is necessary for maintaining mitochondrial network morphology and physiological functions. Mitochondrial fission and fusion were first described in yeast, and found that it is mediated by a group of large GTPases. Although their precise mechanism of action is unclear, the conservation of these GTPases across species suggests that there are some similar mechanisms of mitochondrial dynamics between mammalian cells and yeast. In addition to these GTPases, other proteins have been implicated in the regulation of mitochondrial fusion and fission in mammalian cells, however, almost all of them participate in the regulation of mitochondrial dynamics by interacting with these GTPases. For example, mitochondria-associated PTENinduced kinase 1, a protein linked to familial PD, regulates mitochondrial dynamics through interaction with the fission/fusion machinery.