cells except min6  (ATCC)

Journal: Experimental & Molecular Medicine

Article Title: Mitochondrial-encoded peptide MOTS-c prevents pancreatic islet cell senescence to delay diabetes

doi: 10.1038/s12276-025-01521-1

Figure Lengend Snippet: a , b Treatment with MOTS-c or scrambled control (10 μM, 24 h) and hydrogen peroxide (H 2 O 2 , 200 μM, 24 h) in pancreatic islet cells (pooled from two mice per sample) isolated from littermates of 60-week-old C57BL/6 mice ( n = 3 per sample) led to metabolic changes, as shown in the PCA graph ( a ) and the heat map ( b ). c Enrichment analyses of metabolites were performed for control versus MOTS-c and for H 2 O 2 versus H 2 O 2 + MOTS-c. d A diagram depicting the enriched genes and metabolites analyzed in pancreatic islet cells treated with or without MOTS-c and H 2 O 2 (200 μM, 24 h). e A Venn diagram analysis was performed to find shared pathways by comparing these two enrichment analyses. f Min6 cells overexpressing either empty vector or MOTS-c were treated with glutamine and the expression of genes Slc1a5 , Slc1a5 variant, Gls1/2 and Cd38 , and Cdkn1a and Cdkn2a were assessed. Two-way ANOVA; the error bars are the s.e.m. * P < 0.05, ** P < 0.01 for difference between empty-vector transfected; ## P < 0.01 for difference between empty-vector transfected treated with 5 mM glutamine and MOTS-c transfected treated with 5 mM glutamine. g Min6 cells overexpressing either an empty vector or MOTS-c were analyzed for protein levels of IGF1R, P16, mito-MOTS-c, nuclear MOTS-c- and mTORC1-related molecules and Gls1 in the presence or absence of glutamine (5 mM), H 2 O 2 (200 μM, 24 h) or both.

Article Snippet: All cells except Min6 (ref. ) were purchased from ATCC.

Techniques: Control, Isolation, Plasmid Preparation, Expressing, Variant Assay, Transfection

Journal: Experimental & Molecular Medicine

Article Title: Mitochondrial-encoded peptide MOTS-c prevents pancreatic islet cell senescence to delay diabetes

doi: 10.1038/s12276-025-01521-1

Figure Lengend Snippet: a , b MOTS-c or scrambled ex vivo treatment (10 μM, 24 h) was applied to pancreatic islet cells isolated from four littermates of 60-week-old C57BL/6 mice ( n = 2 per group) to analyze transcriptional changes; these changes were assessed using a PCA plot analyzed by using the scikit-learn Python package ( a ) and a hierarchical heat map ( b ). c – f KEGG and GO analyses (adjusted P value <0.05) indicated that the affected genes are associated with metabolism, cellular communication and signaling and transport ( c ); analysis included: Gene Ontology: biological process (GO: BP) ( d ) Gene Ontology: cellular components (GO: CC) ( e ) Gene Ontology: molecular function (GO: MF) ( f ) were analyzed. g , h The upset plots were used to identify intersecting sets, which are commonly shared genes related to metabolism (pink), signaling (orange) and transport (green); these commonly shared genes, categorized as either upregulated ( g ) or downregulated ( h ) (blue), were displayed in a heat map. i , MOTS-c or scrambled ex vivo treatment (10 μM, 24 h) was applied to pancreatic islet cells isolated from littermates of 60-week-old C57BL/6 mice ( n = 3 per group). j pLJM1-MOTS-c or pLJM1-empty vectors were overexpressed in Min6 cells. Then, cells were treated with or without hydrogen peroxide (200 μM, 24 h) for senescence induction. Subsequently, the expression of genes involved in aspartate–glutamate pathway ( Mdh1 , Mdh1b , Mdh2 , Got1 and Got2 ), EphA5-ephrina5 genes and senescence-related genes ( Cd38 and Grem1 ) were analyzed in both sets.

Article Snippet: All cells except Min6 (ref. ) were purchased from ATCC.

Techniques: Ex Vivo, Isolation, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Mitochondrial-encoded peptide MOTS-c prevents pancreatic islet cell senescence to delay diabetes

doi: 10.1038/s12276-025-01521-1

Figure Lengend Snippet: a Publicly available datasets ( GSE137027 , GSE64553 , GSE72815 , GSE98440 and GSE102004 ) were analyzed for mtRNR1 (MOTS-c) mRNA expression levels (Supplementary Table ). b To explore the underlying mechanism of MOTS-c regulation in senescence, actinonin (50 μM, 24 h) was used to specifically deplete mtDNA in pancreatic islet cells isolated from 12-week-old C57BL/6 mice. c , Min6 cells were transfected with pGenLenti-empty or pGenLenti-Cdkn2a vectors. In b and c the senescence markers (Igf1r, P16INK4a and γ-H2AX) and mTORC1 pathway-related (p-mTOR-2448, p-p70S6K and p-4EBP-1) proteins were analyzed. The pancreatic islet cells from 12- and 90-week-old mice were treated with either hydrogen peroxide (200 μM, 24 h) and doxorubicin (200 nM, 24 h). d The β-gal + p21 + population in pancreatic islets were analyzed. Two-way ANOVA; the error bars are the s.e.m. * P < 0.05, **** P < 0.0001 for comparison. e Hydrogen peroxide and doxorubicin were treated in pancreatic islet cells isolated from 12- or 90-week-old mice to analyze MOTS-c levels. All western blot data are representative of at least three independent experiments. f Treatment with MOTS-c (10 μM, 24 h), with or without H 2 O 2 (200 μM, 24 h), in pancreatic islet cells isolated from 12-week-old C57BL/6 mice prevented senescence markers, including Cdkn1a , Cdkn2a , Cxcl10 and Il-1b mRNA levels. Two-way ANOVA; the error bars are the s.e.m. * P < 0.05, ** P < 0.01, **** P < 0.0001 for comparison. g Pancreatic islet cells isolated from 12-week-old C57BL/6 mice were treated with H 2 O 2 to analyze protein expression levels of γ-H2AX and P16 INK4A . Housekeeping mitochondrial and cytoplasmic proteins (MTCOII and β-actin) were confirmed by western blot. h Treatment with MOTS-c (10 μM, 24 h) in the presence or absence of H 2 O 2 (200 μM, 24 h) in pancreatic islet cells isolated from 12-week-old C57BL/6 mice ( n = 5 per group) was followed by staining and analysis for β-gal, IL-1β, Cxcl10, IL-6 and Igf1r using flow cytometry. Two-way ANOVA; the error bars are the s.e.m. **** P < 0.0001 for comparison.

Article Snippet: All cells except Min6 (ref. ) were purchased from ATCC.

Techniques: Expressing, Isolation, Transfection, Comparison, Western Blot, Staining, Flow Cytometry