The expression of melanocytic markers in undifferentiated and differentiated ADSCs detected by immunofluorescence microscopy.

The expression levels of HMB45, MITF, MelEM, Mel2, MelanA, MATP, and tyrosinase (TYR) in undifferentiated and differentiated ADSCs, as well as NHEMs, were examined by immunofluorescence microscopy. All melanocytic markers except for TYR were detected.</p

siRNA-mediated downregulation of HMB45 in differentiated ADSCs.

siRNA resulted in the downregulation of HMB45 in undifferentiated ADSCs and differentiated ADSCs.</p

The expression of HMB45 and MITF in Mel2-positive and Mel2-negative cells.

The expression of HMB45 and MITF in Mel2-positive cells was increased compared to that in Mel2-negative cells, as shown by immunofluorescence microscopy (A). The expression of HMB45 and MITF in Mel2-positive cells was increased compared to that in Mel2-negative cells, as shown by RT-PCR (B). There was almost no difference in the expression of KIT in Mel2-positive cells and Mel2-negative cells, as shown by RT-PCR (B). The expression of MITF in Mel2-positive cells was increased compared to that in Mel2-negative cells, as shown by western blot analysis (C).</p

The expression of melanocytic markers in undifferentiated and differentiated ADSCs detected by RT-PCR.

The expression levels of HMB45, MITF, PAX3, and KIT in undifferentiated ADSCs and differentiated ADSCs were compared with those in melanocytes by RT-PCR. The expression levels of HMB45, MITF, PAX3, and KIT were higher in differentiated ADSCs than in undifferentiated ADSCs.</p

Immunofluorescence and immunoelectron microscopy with an anti-HMB45 antibody in undifferentiated ADSCs, differentiated ADSCs and melanoma cells.

Note that the immunostaining intensity was increased in differentiated ADSCs. Immunogold particles mainly reacted with tubular structures due to the lack of mature melanosomes.</p

The expression of HMB45 and MITF in undifferentiated and differentiated ADSCs by western blotting.

HMB45 expression in undifferentiated and differentiated ADSCs was detected by western blotting (A). MITF expression in undifferentiated and differentiated ADSCs was detected by western blotting (B). The expression of MITF was increased in differentiated ADSCs.</p

Three-dimensional epidermal culture using normal human epidermal keratinocytes (NHEKs) and normal human epidermal melanocytes (NHEMs) and NHEKs and differentiated ADSCs.

HMB45- and tyrosinase (TYR)-positive cells were observed in the basal layer of both epidermis-like structures (A). Melanin deposits were induced by UVB irradiation in 3D epidermal culture using NHEKs and NHEMs, as well as in NHEKs and differentiated ADSCs and were detected by Fontana-Masson staining (B).</p

Pigmented cells were detected among differentiated ADSCs in an L-DOPA reaction assay.

An L-DOPA reaction assay was performed on undifferentiated ADSCs, differentiated ADSCs, NHEMs, and Mel2-positive and Mel2-negative cells. Pigmented L-DOPA-positive cells were observed among differentiated ADSCs and NHEMs (A). The L-DOPA reaction was not significantly different in Mel2-positive cells and Mel2-negative cells (B).</p

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Image1_Reduced ER-mitochondrial contact sites and mitochondrial Ca2+ flux in PRKN-mutant patient tyrosine hydroxylase reporter iPSC lines.TIF

Endoplasmic reticulum-mitochondrial contact sites (ERMCS) play an important role in mitochondrial dynamics, calcium signaling, and autophagy. Disruption of the ERMCS has been linked to several neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). However, the etiological role of ERMCS in these diseases remains unclear. We previously established tyrosine hydroxylase reporter (TH-GFP) iPSC lines from a PD patient with a PRKN mutation to perform correlative light-electron microscopy (CLEM) analysis and live cell imaging in GFP-expressing dopaminergic neurons. Here, we analyzed ERMCS in GFP-expressing PRKN-mutant dopaminergic neurons from patients using CLEM and a proximity ligation assay (PLA). The PLA showed that the ERMCS were significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control under normal conditions. The reduction of the ERMCS in PRKN-mutant patient dopaminergic neurons was further enhanced by treatment with a mitochondrial uncoupler. In addition, mitochondrial calcium imaging showed that mitochondrial Ca2+ flux was significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control. These results suggest a defect in calcium flux from ER to mitochondria is due to the decreased ERMCS in PRKN-mutant patient dopaminergic neurons. Our study of ERMCS using TH-GFP iPSC lines would contribute to further understanding of the mechanisms of dopaminergic neuron degeneration in patients with PRKN mutations.</p