Supplementary Files

<strong>Evaluating SH-SY5Y cells as a dopaminergic neuronal model: morphological, transcriptomic, and proteomic insights </strong>

<strong>Background/aim:</strong> The SH-SY5Y neuroblastoma cell line is a popular <em>in vitro</em> model for neurodegenerative disease research, especially Parkinson's Disease (PD) research, but its use is complicated by limitations like the the persistence of neuroblastoma-like features, unstable differentiation, mitochondrial dysfunction and cellular stress. To address these limitations, a blended, nine day differentiation protocol using RA, BDNF, and dbcAMP sequentially was tested in this study. By evaluating key neuronal, dopaminergic, and PD-related markers, the research aims to determine if these differentiated SH-SY5Y cells are a suitable model for studying PD.

<strong>Materials and methods:</strong> A blended differentiation protocol using RA, BDNF, and dbcAMP was applied to SH-SY5Y cells. Morphological changes were evaluated by Immunofluorescence microscopy (IF). Furthermore, mostly dopaminergic neuronal markers associated with PD were used for characterization purposes. nLC-MS/MS proteome analysis was performed to identify changes in protein expression related to differentiation.

<strong>Results:</strong> Differentiation led to neuron-like morphology with extended neurites. Gene expression analyses revealed upregulation of several neuronal markers, such as Nestin and MAP2, indicating progression from progenitor to neuron-like states. Furthermore, some dopaminergic markers, such as TH and Nurr1, showed elevated expression with asynchronous expression patterns suggesting heterogeneity in the differentiation process. Proteomic analysis indicated significant changes in cell differentiation and neurogenesis. Transient expression of key neuronal markers was observed. The cells required continuous external stimuli.<strong> </strong>

<strong>Conclusion:</strong> While SH-SY5Y cells exhibited dopaminergic characteristics following the blended differentiation protocol, the transient expression of key neuronal markers and the need for continuous external stimuli raised concerns about the stability and functional maturity of these differentiated cells as an <em>in vitro</em> PD model. These findings suggest that SH-SY5Y cells might not fully capture the properties of mature neurons.