Fig. 4: MT promotes mitofusin2 (MFN2)-mediated neurodifferentiation potential of hiPSCs. | Cell Death Discovery

Fig. 4: MT promotes mitofusin2 (MFN2)-mediated neurodifferentiation potential of hiPSCs.

From: Melatonin orchestrates mitochondrial fusion dynamics-mediated WNT/β-catenin signaling to promote dopaminergic neuronal differentiation of human iPS and nerve regeneration in a MPTP-induced mouse model of Parkinson’s disease

Fig. 4

A Expression of mitochondrial fusion and fission-related proteins MFN1 (86KDa), MFN2 (88KDa), Fis1 (17KDa), DRP1 (1:1000) (80KDa), OPA1 (1:1000)(80-100KDa) and MFF (28-35KDa) was determined by western blot after 7 days of MT and Luzindole treatment in NSCs. β-Actin(42KDa) as the control group. B, C Transmission electron microscopy is used for statistical analysis of the transition of mitochondrial morphology after 7 days of MT and Luzindole treatment in NSCs. Mitochondria with a length less than 0.5 micrometers are defined as fragmented, those longer than 1 micrometer are elongated, and those in the middle are intermediate. Scale bars, 1 μm. D, E MitoTracker Red staining corroborated MT’s role in mitochondrial morphological transformation (fragmented, intermediate, elongated) through MT1/2 modulation. Scale bars (Normal), 20 μm; Scale bars (Amplified), 10 μm. F Expression of MFN2(88KDa) at the protein level was verified by Western blot. β-Actin(42KDa) as the control group. (G) Transmission electron microscopy analyzes the transition of mitochondrial morphology of MFN2 overexpressing and knockdown-hiPSCs. Scale bars, 1 μm. H The morphology of differentiated neurons derived from MFN2 overexpressing and knockdown-hiPSCs. Scale bars, 20 μm. I The statistics of number of branches per NF in the differentiated neurons. J, K Immunofluorescence staining of neuron markers (NFL, NeuN) and the differentiation efficiency statistics. Scale bars, 50 μm. L Immunofluorescence staining and quantitative analysis of neuronal aggregations. White arrows, neuronal aggregation. Scale bars, 10 μm. M The morphology of differentiated DA neurons derived from MFN2 overexpressing and knockdown-hiPSCs. Scale bars, 20 μm. N The statistics of number of branches per NF in directed differentiated DA neurons. O–Q Immunofluorescence staining of DA neuron markers (TH, EN1, NFL, NeuN) and the differentiation efficiency statistics. The efficiency of neuronal differentiation is demonstrated by the positive proportion of neuronal markers (such as the ratio of cells stained positively for EN1 in the cell bodies to the original inoculated cells. Scale bars, 50 μm. R Immunofluorescence staining and quantitative analysis of DA neuronal aggregations. White arrows, neuronal aggregation. Scale bars, 10 μm. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NC (hiPSC)/RC MFN2 NC. #P < 0.05, ##P < 0.01 vs. NC (NSC). &P < 0.05, & &P < 0.01 vs. NSC MT 10 μM.

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