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( a ) TH and DAT staining in matured iPSC-derived dopaminergic neurons used to assess the efficiency of dopaminergic neuron generation. Quantification of Tuj1-positive neurons divided by the total number of cells (DAPI: 4,6-diamidino-2-phenylindole) yielded the percentage of neuronal cells per imaging field. Scale bar = 20 µm. Additionally, the quantification of TH-positive neurons divided by the total number of neurons (number of Tuj1-positive) yielded the percentage of dopaminergic neurons per imaging field. ( b ) Parkin upregulation in healthy iPSC-derived dopaminergic neurons following 48 h of <t>incubation</t> with 4 µM and 8 µM FB231 or DMSO (Cont). GAPDH was used as a loading control. ( c ) FB231 (4 µM) and DMSO-treated dopaminergic neurons were harvested 36 hours after treatment for Western blot and other experiments. ( d and e ) qPCR analysis of the samples used in c . ( f ) Analysis pipeline used for detecting and quantifying organelle morphology. Scale bar = 10 µm. ( g ) Transmission electron microscopy (TEM) characterization of recombinant PFFs before and after sonication, via fibril length. ( h ) Large-field Mitotracker (Mito) and Lysotracker (Lyso) staining and colocalization in neurons previously exposed to PFFs at 1 µg/mL for 24 h, then exposed to 4µM FB231 or DMSO for 24 h. Scale bar = 20 µm.
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( a ) TH and DAT staining in matured iPSC-derived dopaminergic neurons used to assess the efficiency of dopaminergic neuron generation. Quantification of Tuj1-positive neurons divided by the total number of cells (DAPI: 4,6-diamidino-2-phenylindole) yielded the percentage of neuronal cells per imaging field. Scale bar = 20 µm. Additionally, the quantification of TH-positive neurons divided by the total number of neurons (number of Tuj1-positive) yielded the percentage of dopaminergic neurons per imaging field. ( b ) Parkin upregulation in healthy iPSC-derived dopaminergic neurons following 48 h of incubation with 4 µM and 8 µM FB231 or DMSO (Cont). GAPDH was used as a loading control. ( c ) FB231 (4 µM) and DMSO-treated dopaminergic neurons were harvested 36 hours after treatment for Western blot and other experiments. ( d and e ) qPCR analysis of the samples used in c . ( f ) Analysis pipeline used for detecting and quantifying organelle morphology. Scale bar = 10 µm. ( g ) Transmission electron microscopy (TEM) characterization of recombinant PFFs before and after sonication, via fibril length. ( h ) Large-field Mitotracker (Mito) and Lysotracker (Lyso) staining and colocalization in neurons previously exposed to PFFs at 1 µg/mL for 24 h, then exposed to 4µM FB231 or DMSO for 24 h. Scale bar = 20 µm.

Journal: bioRxiv

Article Title: Neural cell state modulation by PARK2 and dopaminergic neuroprotection by small molecule Parkin agonism

doi: 10.64898/2026.04.01.715918

Figure Lengend Snippet: ( a ) TH and DAT staining in matured iPSC-derived dopaminergic neurons used to assess the efficiency of dopaminergic neuron generation. Quantification of Tuj1-positive neurons divided by the total number of cells (DAPI: 4,6-diamidino-2-phenylindole) yielded the percentage of neuronal cells per imaging field. Scale bar = 20 µm. Additionally, the quantification of TH-positive neurons divided by the total number of neurons (number of Tuj1-positive) yielded the percentage of dopaminergic neurons per imaging field. ( b ) Parkin upregulation in healthy iPSC-derived dopaminergic neurons following 48 h of incubation with 4 µM and 8 µM FB231 or DMSO (Cont). GAPDH was used as a loading control. ( c ) FB231 (4 µM) and DMSO-treated dopaminergic neurons were harvested 36 hours after treatment for Western blot and other experiments. ( d and e ) qPCR analysis of the samples used in c . ( f ) Analysis pipeline used for detecting and quantifying organelle morphology. Scale bar = 10 µm. ( g ) Transmission electron microscopy (TEM) characterization of recombinant PFFs before and after sonication, via fibril length. ( h ) Large-field Mitotracker (Mito) and Lysotracker (Lyso) staining and colocalization in neurons previously exposed to PFFs at 1 µg/mL for 24 h, then exposed to 4µM FB231 or DMSO for 24 h. Scale bar = 20 µm.

Article Snippet: Membranes were incubated with primary antibodies overnight at 4°C following blocking with 5% Bovine Serum Albumin (Millipore Sigma, A9647-100G) in TBS with Tween (Thermo Scientific Chemicals, J77500.K2) for 1 h. Primary antibody incubation was done overnight, while secondary antibody incubation was done at room temperature for 1 h. Membranes were imaged using ChemiDoc (Bio-Rad) and analyzed using Image Lab software (Bio-Rad).

Techniques: Staining, Derivative Assay, Imaging, Incubation, Control, Western Blot, Transmission Assay, Electron Microscopy, Recombinant, Sonication