Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of mitochondrial dynamics and function by melatonin type 1 receptor in parkinson’s disease

doi: 10.1007/s00018-025-05995-0

Figure Lengend Snippet: MT1-KD caused mitochondrial dysfunction in SH-SY5Y cells. ( A ) Representative blots of MT1 from control and MT1-KD SH-SY5Y cells. ( B ) Quantification of MT1 protein levels from western blotting ( n =5). ( C ) Statistical analysis of MT1 mRNA levels from control or MT1-KD SH-SY5Y cells ( n =5). ( D ) Statistical analysis of ATP levels after MT1 knockdown ( n =6). ( E ) Representative inverted microscopy images of living cell TMRE staining after MT1 knockdown (10× magnification) ( n =6). Scale bar, 20 μm. ( F ) Statistical analysis of TMRE fluorescent intensity. ( G ) SH-SY5Y cells were treated with 5 μM MT1 inhibitor S26131 for 24 h and subjected to ROS detection using flow cytometry. Representative flow cytometry of staining cells with or without DCF fluorescence. ( H ) Statistical analysis of ROS levels indicated by DCF fluorescence intensity after S26131 treatment ( n =6). Unpaired t -test analysis, * P <0.05, ** P <0.01,*** P <0.001

Article Snippet: SH-SY5Y cells were treated with 5 μM MT1 antagonist S26131 (MedChemExpress, USA) for 24 h and subjected to ROS evaluation (Beyotime).

Techniques: Control, Western Blot, Knockdown, Inverted Microscopy, Staining, Flow Cytometry, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of mitochondrial dynamics and function by melatonin type 1 receptor in parkinson’s disease

doi: 10.1007/s00018-025-05995-0

Figure Lengend Snippet: MT1-KD impaired mitochondrial morphology in SH-SY5Y cells. ( A ) Representative confocal image of MitoTracker-labeled mitochondria in control and MT1-KD SH-SY5Y living cells by (100× magnification). Scale bar, 10 μm. ( B ) Representative TEM micrographs of mitochondria from control and MT1-KD SH-SY5Y cells. Scale bar, 500 nm. ( C ) Statistical analysis of mitochondrial aspect ratio (n=6). ( D ) Statistical analysis of mitochondrial form factor (n=6).. ( E ) Statistical analysis of mitochondrial length ( n =6). ( F ) Statistical analysis of mitochondria–ER contact sites ( n =6). Four pictures of each independent experiment were analyzed. Unpaired t -test analysis, * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: SH-SY5Y cells were treated with 5 μM MT1 antagonist S26131 (MedChemExpress, USA) for 24 h and subjected to ROS evaluation (Beyotime).

Techniques: Labeling, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of mitochondrial dynamics and function by melatonin type 1 receptor in parkinson’s disease

doi: 10.1007/s00018-025-05995-0

Figure Lengend Snippet: MT1-KD increased mitochondrial fission through DRP1 phosphorylation and mitophagy in SH-SY5Y cells. ( A ) Representative blots of OPA1, MFN1, MFN2 and DRP1 protein expression from control or MT1-KD SH-SY5Y cells. ( B – E ) Quantification of OPA1, MFN1, MFN2 and DRP1 protein levels, respectively ( n =5). ( F ) Representative blots of S637-DRP1, pPKA, S616-DRP1, pERK1/2 and ERK1/2 protein expression in control or MT1-KD SH-SY5Y cells. ( G – J ) Quantification of S637-DRP1, pPKA, S616-DRP1, pERK1/2 and ERK1/2 phosphorylation, respectively ( n =5). (K) Representative blots of PINK1, Parkin, P62 and LC3 protein expression from control or MT1-KD SH-SY5Y cells. (L–O) Quantification of PINK1, Parkin, P62 and LC3, respectively ( n =5). Two identical lanes from representative blots in each group represented two repeated experiments. Unpaired t -test analysis, * P <0.05,** P <0.01

Article Snippet: SH-SY5Y cells were treated with 5 μM MT1 antagonist S26131 (MedChemExpress, USA) for 24 h and subjected to ROS evaluation (Beyotime).

Techniques: Phospho-proteomics, Expressing, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of mitochondrial dynamics and function by melatonin type 1 receptor in parkinson’s disease

doi: 10.1007/s00018-025-05995-0

Figure Lengend Snippet: MT1-KO impaired mitochondrial morphology and aggravated mitochondrial fission in mouse primary neurons treated with MPP+. ( A ) Representative TEM micrograph of mitochondria from substantia nigra in WT or MT1-KO transgenic mice aged 4 or 12 months. Scale bar, 500 nm. ( B ) Statistical analysis of mitochondrial length by TEM. Five pictures of each independent experiment and three mitochondria of each picture were analyzed ( n =5). ( C ) Representative immunostaining images of mitochondria with TOM20 and axons with Tuj1 from WT or MT1-KO primary neurons with or without MPP + . Scale bar, 1 μm. ( D ) Statistical analysis of mitochondrial length by immunostaining ( n =5). Two-way ANOVA analysis, * P <0.05,** P <0.01

Article Snippet: SH-SY5Y cells were treated with 5 μM MT1 antagonist S26131 (MedChemExpress, USA) for 24 h and subjected to ROS evaluation (Beyotime).

Techniques: Transgenic Assay, Immunostaining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of mitochondrial dynamics and function by melatonin type 1 receptor in parkinson’s disease

doi: 10.1007/s00018-025-05995-0

Figure Lengend Snippet: MT1-KO aggravated mitochondrial fission through DRP1 dephosphorylation and mitophagy in MPTP-induced mouse model. ( A ) Histogram showing the results of mouse latency to fall during the rotarod test ( n =6). ( B ) Representative blots of TH protein expression from WT or MT1-KO mice with or without MPTP. ( C ) Quantification of TH ( n =5). ( D ) Representative blots of OPA1, MFN1, MFN2 and DRP1 protein expression from WT or MT1-KO mice with or without MPTP. ( E – H ) Quantification of OPA1, MFN1, MFN2 and DRP1 protein levels ( n =5). ( I ) Representative blots of S637-DRP1, pPKA, S616-DRP1, pERK1/2 and ERK1/2 protein expression from WT or MT1-KO mice with or without MPTP. ( J – M ) Quantification of S637-DRP1, pPKA, S616-DRP1, pERK1/2 and ERK1/2 ( n =5). ( N ) Representative blots of P62 and LC3 protein expression from WT or MT1-KO mice with or without MPTP. ( O , P ) Quantification of P62 and LC3, respectively ( n =5). Two-way ANOVA analysis, * P <0.05, ** P <0.01. ns, not significant

Article Snippet: SH-SY5Y cells were treated with 5 μM MT1 antagonist S26131 (MedChemExpress, USA) for 24 h and subjected to ROS evaluation (Beyotime).

Techniques: De-Phosphorylation Assay, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of mitochondrial dynamics and function by melatonin type 1 receptor in parkinson’s disease

doi: 10.1007/s00018-025-05995-0

Figure Lengend Snippet: MT1 deficiency enhanced PFF-induced autophagy inhibition and α-syn aggregation. Neonatal mouse primary cortical neurons were treated with PFFs (1 μg/ml) for 10 days and protein was extracted and subjected to western blotting. ( A ) Representative blots of P62 and LC3 from three repeat experiments. ( B ) Representative blots of α-syn in soluble fraction. ( C ) Representative blots of α-syn in pellet fraction. ( D – G ) Quantification of protein levels of P62, LC3, α-syn monomers and high molecular weight (HMW) α-syn ( n =5). Two-way ANOVA analysis, * P <0.05, ** P <0.01, *** P <0.001. ns, not significant

Article Snippet: SH-SY5Y cells were treated with 5 μM MT1 antagonist S26131 (MedChemExpress, USA) for 24 h and subjected to ROS evaluation (Beyotime).

Techniques: Inhibition, Western Blot, High Molecular Weight

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of mitochondrial dynamics and function by melatonin type 1 receptor in parkinson’s disease

doi: 10.1007/s00018-025-05995-0

Figure Lengend Snippet: MT1 overexpression increased mitochondrial fusion and facilitated autophagic flux to promote α-syn degradation in HEK293T cells. ( A ) HEK293T cells were transfected with PCDNA3.1 or hMT1-Flag and cell lysate was subjected to western blotting. Representative blots of MFN1, MFN2 and DRP1 from three repeat experiments. ( B – D ) Statistical analysis of MFN1, MFN2 and DRP1 protein levels ( n =6). Unpaired t -test. ( E ) Representative blots of P62 and LC3 from three repeat experiments. ( F , G ) Statistical analysis of P62 and LC3II protein levels ( n =6). One-way ANOVA. ( H ) HEK293T cells were transfected with PCDNA3.1 or hMT1-Flag for 48 h; 200 nm BafA1 was added 16 h before protein detection and cell lysate was subjected to western blotting. Representative blots of LC3 from three repeat experiments. ( I ) Statistical analysis of LC3II protein levels ( n =3). Kruskal–Wallis test. ( J ) HEK293T cells were transfected with or without hMT1-Flag for 48 h and infected with mCherry-LC3-EGFP lentivirus for 36 h; 200 nm BafA1 was added 16 h before confocal analysis. Quantification of autophagosomes ( K ) and autolysosomes ( L ) per cell using Image J ( n =5). Scale bar, 5 μm. Two-way ANOVA. ( M ) α-syn-GFP HEK293T stable cell line was transfected with PCDNA3.1 or hMT1-Flag plasmids. Representative blots of P62, LC3 and α-syn-GFP. ( N – P ) Statistical analysis of P62, LC3II and α-syn-GFP protein levels ( n =5). One-way ANOVA. Three identical lanes from representative blots in each group represented three repeat experiments. * P <0.05, ** P <0.01,*** P <0.001

Article Snippet: SH-SY5Y cells were treated with 5 μM MT1 antagonist S26131 (MedChemExpress, USA) for 24 h and subjected to ROS evaluation (Beyotime).

Techniques: Over Expression, Transfection, Western Blot, Infection, Stable Transfection