Characterization of hiPSC-derived dopaminergic neurons in control and VPS13C KD condition. (A) Table with information on the hiPSC lines obtained from Northwestern University Biorepository and Coriell Institute. (B) Schematic of the differentiation process from hiPSCs to dopaminergic neurons and treatment with lentiviral shRNA (14 days) for a non-targeting control and shRNA specifically targeting VPS13C at day 56 with MOI 2. Neurons were harvested on day 70 for the assessment of VPS13C KD efficiency and downstream readouts. Schematic was generated with http://BioRender.com. (C) Representative confocal images from fixed iPSC-derived dopaminergic control and VPS13C KD neurons showing immunostaining of TH (green), βIII-tubulin (magenta), and DAPI (blue) (scale bar: 20 µm). (D) Quantification of the percentage of TH-positive neurons (N = 3). (E and F) (E) Representative immunoblot of TH protein levels in control and VPS13C KD neurons and (F) relative quantification of TH levels. (G–J) Representative immunoblots and relative quantifications of neuronal synaptic marker VMAT2 (* unspecific protein band), neuronal marker βIII-tubulin, and GAPDH with relative quantifications (N = 3). (K) Representative confocal images of immunostaining for endogenous LAMP1 (magenta), TH (green), and DAPI (blue) in control neurons showing smaller LAMP1-positive vesicles (white arrow) in comparison to VPS13C KD neurons showing enlarged and clustered LAMP1-positive vesicles (yellow arrow) (D70) (scale bar: 10 µm, inset: 1 µm). Data represented as mean ± SEM; unpaired two-tailed t test (D, F, H, I, and J); ns: not significant (H, I, and J), *P < 0.05 (D and F). Source data are available for this figure: SourceData FS1.