min6 cells  (AddexBio Inc)

Journal: bioRxiv

Article Title: Genetic background influences the phenotypic penetrance by MAFA S64F MODY in male mice

doi: 10.1101/2025.05.20.653758

Figure Lengend Snippet: A. DEGs identified uniquely in Male Het islets from a mixed background (1238 + 1114 = 2352 genes) versus those uniquely from a C57 background (228 + 855 = 1083 genes) were then overlayed with peaks identified by endogenous MafA CUT&RUN in mouse MIN6 cells (n=11403 peaks). Of these, 250 genes uniquely enriched in a C57 background overlapped with a MafA CUT&RUN peak, while 1210 were uniquely enriched in a Mixed background overlapped with a MafA CUT&RUN peak. B. UCSC Genome Browser tracks showing genomic regions associated with endogenous MafA CUT&RUN peaks near known targets Ins1, Ins2, MafB, and Pdx1, and candidate genes Onecut1, Cry2, Per1, and Per2; MafA CUT&RUN enriched peaks are highlighted in dashed boxes, and regulated genes are depicted below IgG control tracks.

Article Snippet: CUT&RUN was performed on 500,000 dispersed mouse MIN6 cells per condition using CUTANA ChIC/CUT&RUN protocol v3.1 (Epicypher).

Techniques: Control

Journal: bioRxiv

Article Title: Genetic background influences the phenotypic penetrance by MAFA S64F MODY in male mice

doi: 10.1101/2025.05.20.653758

Figure Lengend Snippet: A-B. Immunostaining for MafA show poor detection in Mixed background male Het islets (left) but intact MafA in C57 male Het islets (right). Islets from MafA Λβ included as a negative control. Scale bar, 50μm. C. Left lanes, Western blotting on MIN6 nuclear extract transfected to express either MAFA WT or MAFA S64F shows faster migration in mutant MAFA due to impaired posttranslational modification by phosphorylation. Right lanes, Isolated mouse islets from each genotype and background showed detectable levels of phosphorylated (active) MAFA in C57 background, but relative uniformity of MAFA species with impaired phosphorylation in the Mixed background. D. Quantification of Western blotting bands by Line scan analysis shows greater proportion of phosphorylated MafA species (gray) in Het male islets from the C57 background compared to the Mixed background.

Article Snippet: CUT&RUN was performed on 500,000 dispersed mouse MIN6 cells per condition using CUTANA ChIC/CUT&RUN protocol v3.1 (Epicypher).

Techniques: Immunostaining, Negative Control, Western Blot, Transfection, Migration, Mutagenesis, Modification, Phospho-proteomics, Isolation

Journal: Aging Cell

Article Title: Small Extracellular Vesicles From Human Amniotic Membrane Mesenchymal Stem Cells Rejuvenate Senescent β Cells and Cure Age‐Related Diabetes in Mice

doi: 10.1111/acel.70327

Figure Lengend Snippet: hAMSC‐sEVs ameliorate β‐cell senescence in vitro. (a–d) sEV intervention in H 2 O 2 ‐induced senescence in MIN6 cells. (a) Experimental timeline: cells are pretreated with H 2 O 2 (200 μM, 2 h) with/without sEVs (25–100 ng/μL, 48 h). (b) PKH26‐labeled sEV uptake is shown (red) after 24 h. Scale bars: 100 μm (overview panels); 20μm (oom). (c) Senescence marker staining shows SA‐β‐gal (blue), γ‐H2AX foci (green), and EdU + proliferative cells (red). Scale bars, 50 μm. (d) Quantification shows SA‐β‐gal + cells (%), γ‐H2AX intensity, and EdU + cells (%); n = 5 per group. (e–h) sEV intervention in aging‐associated senescence in C57BL/6J islets from young (2‐month), aged (18‐month), and aged + sEVs (100 ng/μL, 48 h) groups: (e) p16 (red)/insulin (green) co‐staining is shown. Scale bars: 50 μm (overview panels); 10 μm (Zoom). (f) γ‐H2AX (red)/insulin (green) co‐staining is shown. Scale bars: 50 μm (overview panels); 10 μm (Zoom). (g, h) Quantification shows p16 + β‐cells (%) (g) and γ‐H2AX + β‐cells (%) (h); n = 6 per group. (i–k) Molecular profiling. (i) Western blots show senescence markers (Lamin B1, p53, p21, p16). (j) qPCR shows senescence‐related mRNAs ( Cdkn2a, Cdkn1a, Trp53, Lmnb1, Igf1r ); n = 5 per group. (k) qPCR shows SASP mRNAs ( Il1b, Il6, Tnf, Ccl2, Cxcl10, Gdf15, Dusp3, Hsp90aa1 ); n = 5 per group. Each dot represents one independent experiment; data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001; ns, not significant.

Article Snippet: Mitochondrial Ca 2+ dynamics in MIN6 cells (5 × 10 4 cells/cm 2 , poly‐L‐lysine‐coated dishes) were assessed through Rhod‐2 AM‐based confocal imaging following 24 h adhesion and 48–72 h post‐intervention incubation (transfection/sEVs), with cells pre‐equilibrated in 2.8 mM glucose KRBH (37°C/5% CO2, 1.5 h) before 1X Rhod‐2 AM (Cat#S1062M, Beyotime, Shanghai, China) loading (37°C/30 min) and triplicate KRBH washing.

Techniques: In Vitro, Labeling, Marker, Staining, Western Blot

Journal: Aging Cell

Article Title: Small Extracellular Vesicles From Human Amniotic Membrane Mesenchymal Stem Cells Rejuvenate Senescent β Cells and Cure Age‐Related Diabetes in Mice

doi: 10.1111/acel.70327

Figure Lengend Snippet: hAMSC‐sEVs restore insulin secretion and mitochondrial metabolic homeostasis in senescent β‐cells. (a–e) sEV intervention in H 2 O 2 ‐induced senescence in MIN6 cells; cells are pretreated with H 2 O 2 (200 μM, 2 h) with/without sEVs (25–100 ng/μL, 48 h). (a) Insulin immunofluorescence (red) is shown. Scale bars, 50 μm. (b) sEVs dose‐dependently enhance insulin content; n = 5 per group. (c) GSIS profile is restored; n = 5 per group. (d, e) β‐cell maturation markers ( Ins1, Mafa, Pdx1, Slc2a2 ) are upregulated at the mRNA (d) and protein (e) levels. (f–h) Oxygen consumption rate (OCR) analysis (f, g) and ROS levels (h) in MIN6 cells under three conditions: Control (Ctrl), senescent (S), and senescent + sEVs (100 ng/μL; S + sEVs); n = 6 per group. (i) Insulin secretion in C57BL/6J islets from young (2‐month), aged (18‐month), and aged + sEVs (100 ng/μL, 48 h) groups under low (3.3 mM) versus high (16.7 mM) glucose. (j, k) Islet perifusion of islets from 18‐month C57BL/6J mice treated with sEVs (100 ng/μL) or vehicle for 48 h (j); AUC is analyzed across phases: Basal (10–15 min), first phase (15–20 min), and second phase (20–30 min) (k). (l–n) OCR analysis (l, m) and ROS levels (n) in C57BL/6J islets from aged (18‐month) and aged + sEVs (100 ng/μL, 48 h) groups; n = 5–6 per group. Each dot represents one independent experiment; data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Mitochondrial Ca 2+ dynamics in MIN6 cells (5 × 10 4 cells/cm 2 , poly‐L‐lysine‐coated dishes) were assessed through Rhod‐2 AM‐based confocal imaging following 24 h adhesion and 48–72 h post‐intervention incubation (transfection/sEVs), with cells pre‐equilibrated in 2.8 mM glucose KRBH (37°C/5% CO2, 1.5 h) before 1X Rhod‐2 AM (Cat#S1062M, Beyotime, Shanghai, China) loading (37°C/30 min) and triplicate KRBH washing.

Techniques: Immunofluorescence, Control

Journal: Aging Cell

Article Title: Small Extracellular Vesicles From Human Amniotic Membrane Mesenchymal Stem Cells Rejuvenate Senescent β Cells and Cure Age‐Related Diabetes in Mice

doi: 10.1111/acel.70327

Figure Lengend Snippet: hAMSC‐sEV‐miR‐21‐5p targets the IL‐6RA/STAT3 axis to ameliorate β‐cell senescence. (a) Heatmap shows differentially regulated genes (|log₂FC| > 1, p < 0.05) between senescent MIN6 cells (S) and hAMSC‐sEV–treated senescent MIN6 cells (sEVs) by RNA‐seq. (b) KEGG pathway enrichment is performed for genes significantly modulated by hAMSC‐sEVs. (c) Gene set enrichment analysis (GSEA) indicates enrichment for the IL‐6 family cytokine receptor–ligand interaction signature (NES, normalized enrichment score). (d) Venn diagram illustrates the overlap between downregulated DEGs in sEV‐treated senescent MIN6 cells and miR‐21‐5p–predicted targets (TargetScan and miRanda). (e) Western blots show IL‐6RA expression in H₂O₂‐induced senescent MIN6 cells and in islets isolated from young (2‐month) and aged (18‐month) C57BL/6J mice. (f) IL‐6RA protein levels are shown in MIN6 cells transfected with NC mimic, miR‐21‐5p mimic, NC inhibitor, or miR‐21‐5p inhibitor. (g) Schematic shows wild‐type and mutant Il6ra 3′UTR luciferase reporter constructs. (h) Dual‐luciferase assays validate miR‐21‐5p binding to the Il6ra 3′UTR; n = 6 per group. (i) Western blots show IL‐6RA, p‐STAT3 (Tyr705), total STAT3, p21, and PDX1 in Ctrl, S, S + sEVs, and S + 21‐5p mimic MIN6 cells. (j) Representative immunofluorescence images (left) and quantification (right) show pY705‐STAT3 nuclear translocation across groups. Scale bar, 50 μm. Each dot represents one field‐of‐view mean (≈30–50 cells), collected across independent experiments; n = 12 fields per group from 3 independent experiments. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: Mitochondrial Ca 2+ dynamics in MIN6 cells (5 × 10 4 cells/cm 2 , poly‐L‐lysine‐coated dishes) were assessed through Rhod‐2 AM‐based confocal imaging following 24 h adhesion and 48–72 h post‐intervention incubation (transfection/sEVs), with cells pre‐equilibrated in 2.8 mM glucose KRBH (37°C/5% CO2, 1.5 h) before 1X Rhod‐2 AM (Cat#S1062M, Beyotime, Shanghai, China) loading (37°C/30 min) and triplicate KRBH washing.

Techniques: RNA Sequencing, Western Blot, Expressing, Isolation, Transfection, Mutagenesis, Luciferase, Construct, Binding Assay, Immunofluorescence, Translocation Assay

Journal: Aging Cell

Article Title: Small Extracellular Vesicles From Human Amniotic Membrane Mesenchymal Stem Cells Rejuvenate Senescent β Cells and Cure Age‐Related Diabetes in Mice

doi: 10.1111/acel.70327

Figure Lengend Snippet: Integrated multi‐omics profiling reveals STAT3‐mediated transcriptional regulation of Mcu in β‐cell senescence. (a) Heatmaps show CUT&Tag‐seq signals of phosphorylated STAT3 (pY705‐STAT3) around transcription start sites (TSSs) in H₂O₂‐induced senescent MIN6 cells (S_1/S_2) versus normal controls (Ctrl_1/Ctrl_2). (b) KEGG pathway enrichment is shown for genes associated with differential pSTAT3 binding peaks; the top six pathways ranked by significance are listed with genes. (c) De novo motif analysis using HOMER identifies a characteristic pSTAT3 motif; motif significance is indicated by grayscale letter height. (d) Venn diagram illustrates convergence of RNA‐seq differentially expressed genes (DEGs, blue), proteomic differentially expressed proteins (DEPs, red), and CUT&Tag binding peaks (green) in senescent (S) versus control (Ctrl) MIN6 cells. (e) JASPAR‐predicted STAT3 binding motifs are mapped in the Mcu promoter. (f, g) Luciferase assays assess serial 5′ truncations (f) and site‐directed mutants (g) of the Mcu promoter in MIN6 cells after IL‐6 stimulation; n = 6 per group. (h) ChIP‐qPCR validates phosphorylation‐dependent STAT3 occupancy at the Mcu promoter; n = 6 per group. (i) Western blots show MCU, PDX1, p21, and IL‐6 in H₂O₂‐induced senescent MIN6 cells transfected with OE‐ Mcu or OE‐NC for 48 h. Data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Mitochondrial Ca 2+ dynamics in MIN6 cells (5 × 10 4 cells/cm 2 , poly‐L‐lysine‐coated dishes) were assessed through Rhod‐2 AM‐based confocal imaging following 24 h adhesion and 48–72 h post‐intervention incubation (transfection/sEVs), with cells pre‐equilibrated in 2.8 mM glucose KRBH (37°C/5% CO2, 1.5 h) before 1X Rhod‐2 AM (Cat#S1062M, Beyotime, Shanghai, China) loading (37°C/30 min) and triplicate KRBH washing.

Techniques: Biomarker Discovery, Binding Assay, RNA Sequencing, Control, Luciferase, ChIP-qPCR, Phospho-proteomics, Western Blot, Transfection

Journal: Aging Cell

Article Title: Small Extracellular Vesicles From Human Amniotic Membrane Mesenchymal Stem Cells Rejuvenate Senescent β Cells and Cure Age‐Related Diabetes in Mice

doi: 10.1111/acel.70327

Figure Lengend Snippet: MiR‐21‐5p attenuates β‐cell senescence by suppressing the IL‐6RA/STAT3/MCU axis to restore mitochondrial calcium‐redox coupling. (a–i) H₂O₂‐induced senescence model in MIN6 cells (200 μM, 2 h) with combinatorial interventions of miR‐21‐5p mimic and Mcu‐targeting shRNA (shMcu); cells are analyzed 48 h post‐transfection. (a) Representative SA‐β‐gal staining. (b) Quantification of SA‐β‐gal–positive cells for (a); n = 6 per group. Scale bar, 50 μm. (c) Representative co‐staining with MitoSOX (superoxide, green) and MitoTracker (mitochondrial mass, red). Scale bar, 50 μm. (d) Quantification of MitoSOX fluorescence intensity for (c); n = 8 per group. (e) Representative JC‐1 staining (red, high ΔΨm aggregates; green, low ΔΨm monomers). Scale bar, 50 μm. (f) Quantification of red/green ratios (ΔΨm index) for (e); n = 6 per group. (g, h) Mitochondrial Ca 2+ levels ([Ca 2+ ]ₘᵢₜₒ) are measured with Rhod‐2 after stimulation with 20 mM glucose; (g) shows average fluorescence traces, and (h) shows maximal Rhod‐2 signals (normalized to basal); n = 5 per group. (i) Western blots show MCU, IL‐6RA, pY705‐STAT3, total STAT3, p16, p21, and PDX1. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Mitochondrial Ca 2+ dynamics in MIN6 cells (5 × 10 4 cells/cm 2 , poly‐L‐lysine‐coated dishes) were assessed through Rhod‐2 AM‐based confocal imaging following 24 h adhesion and 48–72 h post‐intervention incubation (transfection/sEVs), with cells pre‐equilibrated in 2.8 mM glucose KRBH (37°C/5% CO2, 1.5 h) before 1X Rhod‐2 AM (Cat#S1062M, Beyotime, Shanghai, China) loading (37°C/30 min) and triplicate KRBH washing.

Techniques: shRNA, Transfection, Staining, Fluorescence, Western Blot

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

Journal: Journal of Diabetes

Article Title: Taurine Alleviates Pancreatic β‐Cell Senescence by Inhibition of p53 Pathway

doi: 10.1111/1753-0407.70100

Figure Lengend Snippet: Taurine supplement alleviates doxorubicin‐induced β‐cell inflammation and senescence. MIN6 cells were pre‐treated with 100 μM taurine for 24 h, followed by 200 nM doxorubicin (DOXO) treatment for 24 h. Cells were cultured in FBS‐free medium to avoid possible contamination of taurine. (A) QPCR analysis of the genes related to inflammation, senescence, and apoptosis in each group of doxorubicin‐induced senescence model. ( n = 3) Relative mRNA levels were normalized to β‐actin. (B) Immunoblotting analysis of p53 and p21 and densitometric quantification. ( n = 3). (C) Immunofluorescence staining of DNA damage marker γ–H2AX in each group (scale bar: 100 μm). ( n = 5). (D) FACS analysis of β‐gal+ PI‐(senescent) and PI+ (dead) MIN6 cells. All results are presented as mean ± SEM. Significance was determined using two‐way ANOVA with Tukey correction. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse pancreatic β‐cell line MIN6 (AddexBio Technologies, Cat#C0018008) and rat insulinoma cell INS‐1E (AddexBio Technologies, Cat#C0018009) were cultured in DMEM (Gibco, Cat#12800082) or RPMI 1640 supplemented with 15% FBS, 1% penicillin–streptomycin, and 50 μM β‐mercaptoethanol.

Techniques: Cell Culture, Western Blot, Immunofluorescence, Staining, Marker

Journal: Journal of Diabetes

Article Title: Taurine Alleviates Pancreatic β‐Cell Senescence by Inhibition of p53 Pathway

doi: 10.1111/1753-0407.70100

Figure Lengend Snippet: Taurine supplementation alleviates TNF‐α‐induced β‐cell inflammation and senescence. MIN6 cells were pre‐treated with 100 μM taurine for 24 h, followed by 20 ng/mL TNF‐α treatment for 24 h. Cells were cultured in FBS‐free medium to avoid possible contamination of taurine. (A) QPCR analysis of the genes related to senescence in each group of TNF‐α‐induced senescence model. ( n = 4). Relative mRNA levels were normalized to β‐actin. (B) QPCR analysis of the genes related to inflammation and apoptosis in each group. ( n = 4). Relative mRNA levels were normalized to β‐actin. (C) Immunoblotting analysis of p53 and p21 in each group and densitometric quantification. ( n = 3). All results are presented as mean ± SEM. Significance was determined using two‐way ANOVA with Tukey correction. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse pancreatic β‐cell line MIN6 (AddexBio Technologies, Cat#C0018008) and rat insulinoma cell INS‐1E (AddexBio Technologies, Cat#C0018009) were cultured in DMEM (Gibco, Cat#12800082) or RPMI 1640 supplemented with 15% FBS, 1% penicillin–streptomycin, and 50 μM β‐mercaptoethanol.

Techniques: Cell Culture, Western Blot

Journal: Journal of Diabetes

Article Title: Taurine Alleviates Pancreatic β‐Cell Senescence by Inhibition of p53 Pathway

doi: 10.1111/1753-0407.70100

Figure Lengend Snippet: β‐cells acquire taurine through Slc6a6‐mediated uptake. (A) QPCR analysis of taurine biosynthesis related genes and its transporter Slc6a6 in MIN6 cells and mouse hepatocytes. The results are presented as relative levels over respective gene expression in mouse hepatocytes. ( n = 4). (B, C) MIN6 cells were transfected with siRNA against Scramble or Slc6a6 for 24 h, followed by treatment with taurine (100 μM) or vehicle for 24 h. (B) Immunoblotting analysis of SLC6A6 protein level in each group. ( n = 3). (C) Intracellular taurine levels in the transfected MIN6 cells. ( n = 4). (D) MIN6 cells were pre‐treated with non‐FBS culture medium. The cells were then treated with taurine (100 μM) for 24 h, followed by treatment with SLC6A6 inhibitor (SLC6A6i) (100 μM) or vehicle for 30 min. Intracellular taurine concentration was measured by LC–MS/MS. ( n = 3). All results are presented as mean ± SEM. Significance was determined using two‐tailed independent student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse pancreatic β‐cell line MIN6 (AddexBio Technologies, Cat#C0018008) and rat insulinoma cell INS‐1E (AddexBio Technologies, Cat#C0018009) were cultured in DMEM (Gibco, Cat#12800082) or RPMI 1640 supplemented with 15% FBS, 1% penicillin–streptomycin, and 50 μM β‐mercaptoethanol.

Techniques: Gene Expression, Transfection, Western Blot, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Journal of Diabetes

Article Title: Taurine Alleviates Pancreatic β‐Cell Senescence by Inhibition of p53 Pathway

doi: 10.1111/1753-0407.70100

Figure Lengend Snippet: The protective effects of taurine against β‐cell senescence depend on its transporter SLC6A6. (A, B) MIN6 cells were pre‐treated with the SLC6A6 inhibitor (SLC6A6i) (100 μM) or vehicle for 30 min, followed by treatment with taurine (100 μM) and doxorubicin (200 nM) or vehicle for 24 h in non‐FBS culture medium. The intracellular taurine concentration was then measured by LC–MS/MS. ( n = 3). (B) Immunoblotting analysis of p53 and p21 in each group. (C–F) MIN6 cells were pre‐treated with doxorubicin (200 nM). The cells were then transfected with siRNA against Scramble or Slc6a6 for 24 h, followed by treatment with taurine (100 μM) or vehicle for 24 h. (C) Immunoblotting analysis of SLC6A6, p53, and p21 in each group. ( n = 3). (D) QPCR analysis of gene expressions related to senescence in each group ( n = 4). (E) QPCR analysis of the genes related to β‐cell specific SASP in each group. ( n = 4). (F) QPCR analysis of genes related to inflammation and apoptosis. ( n = 4). All results are presented as mean ± SEM. Significance was determined using two‐way ANOVA with Tukey correction. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse pancreatic β‐cell line MIN6 (AddexBio Technologies, Cat#C0018008) and rat insulinoma cell INS‐1E (AddexBio Technologies, Cat#C0018009) were cultured in DMEM (Gibco, Cat#12800082) or RPMI 1640 supplemented with 15% FBS, 1% penicillin–streptomycin, and 50 μM β‐mercaptoethanol.

Techniques: Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transfection

Journal: Journal of Diabetes

Article Title: Taurine Alleviates Pancreatic β‐Cell Senescence by Inhibition of p53 Pathway

doi: 10.1111/1753-0407.70100

Figure Lengend Snippet: Taurine mitigates senescence, inflammation, and oxidative stress via a p53‐dependent pathway while preserving mitochondrial function independently of p53. (A–C) MIN6 cells were pre‐treated with DOXO (200 nM). The cells were then transfected with siRNA against scramble or p53 for 24 h, followed by treatment with taurine (100 μM) or vehicle for 24 h. Cells were cultured in FBS‐free medium to avoid possible contamination of taurine. (A) QPCR analysis of the genes related to senescence and inflammation in each group. ( n = 4) Relative mRNA levels were normalized to β‐actin. (B) Cellular content of malondialdehyde (MDA) in each group. ( n = 4). (C) Mitochondrial membrane potential was measured using TMRE mitochondrial membrane potential assay. ( n = 7). All results are presented as mean ± SEM. Significance was determined using two‐way ANOVA with Tukey correction. * p < 0.05, ** p < 0.005, *** p < 0.001.

Article Snippet: Mouse pancreatic β‐cell line MIN6 (AddexBio Technologies, Cat#C0018008) and rat insulinoma cell INS‐1E (AddexBio Technologies, Cat#C0018009) were cultured in DMEM (Gibco, Cat#12800082) or RPMI 1640 supplemented with 15% FBS, 1% penicillin–streptomycin, and 50 μM β‐mercaptoethanol.

Techniques: Preserving, Transfection, Cell Culture, Membrane

Journal: Journal of Diabetes

Article Title: Taurine Alleviates Pancreatic β‐Cell Senescence by Inhibition of p53 Pathway

doi: 10.1111/1753-0407.70100

Figure Lengend Snippet: Identification of Taurine‐CDKN2AIP binding in pancreatic β cells. (A, B) Limited proteolysis‐mass spectrometry (LiP‐MS) was used to screen for taurine interacting proteins in the INS1E β‐cell proteome. Heatmap shows potential taurine binding targets identified by LiP‐MS. Vehicle: N = 3. Taurine: N = 3. (B) p53 pathway related proteins levels between two groups and their binding scores with taurine. (C) Three‐dimensional diagram of the binding modes between human CDKN2AIP and taurine. Taurine potentially binds to CDKN2AIP via residues PRO484, LEU485, LYS486. (D) DARTS analysis using MIN6 cell lysates incubated with taurine. (E) DARTS analysis using INS1E cell lysates incubated with taurine. (F) 500 ng of CDKN2AIP recombinant protein were subjected to SDS‐PAGE and silver staining to assess purity. (G) DARTS analysis using CDKN2AIP recombinant protein incubated with taurine. (H) MIN6 cells treated with taurine (100 μM, 24 h) or vehicle were subjected to immunoprecipitation against CDKN2AIP.

Article Snippet: Mouse pancreatic β‐cell line MIN6 (AddexBio Technologies, Cat#C0018008) and rat insulinoma cell INS‐1E (AddexBio Technologies, Cat#C0018009) were cultured in DMEM (Gibco, Cat#12800082) or RPMI 1640 supplemented with 15% FBS, 1% penicillin–streptomycin, and 50 μM β‐mercaptoethanol.

Techniques: Binding Assay, Mass Spectrometry, Incubation, Recombinant, SDS Page, Silver Staining, Immunoprecipitation

Journal: Journal of Diabetes

Article Title: Taurine Alleviates Pancreatic β‐Cell Senescence by Inhibition of p53 Pathway

doi: 10.1111/1753-0407.70100

Figure Lengend Snippet: Taurine treatment accelerates p53 degradation by binding to CDKN2AIP. (A) p53 protein degradation was detected using cycloheximide (CHX, 10 μM) chase assay. (B) HEK 293 cells were transfected with plasmids encoding GFP‐tagged CDKN2AIP (WT) and CDKN2AIP‐triple mutant (MT) for 48 h. DARTS analysis was performed using cell lysates incubated with taurine, followed by immunoblotting analysis as indicated. (C) INS‐1E cells were transfected with plasmids encoding GFP control, GFP‐tagged CDKN2AIP, and its triple mutant for 24 h, followed by taurine treatment for 24 h. Immunoblotting analysis of CDKN2AIP and p53 in each group.

Article Snippet: Mouse pancreatic β‐cell line MIN6 (AddexBio Technologies, Cat#C0018008) and rat insulinoma cell INS‐1E (AddexBio Technologies, Cat#C0018009) were cultured in DMEM (Gibco, Cat#12800082) or RPMI 1640 supplemented with 15% FBS, 1% penicillin–streptomycin, and 50 μM β‐mercaptoethanol.

Techniques: Binding Assay, Transfection, Mutagenesis, Incubation, Western Blot, Control