min6 cells  (Selleck Chemicals)

Journal: Febs Letters

Article Title: Crosstalk between the ribosome quality control‐associated E3 ubiquitin ligases LTN1 and RNF10

doi: 10.1002/1873-3468.70230

Figure Lengend Snippet: Loss of LTN1 leads to upregulation of RNF10 in human and mouse cells. (A) Western blot analysis of E3 ubiquitin‐protein ligase ZNF598 (ZNF598), E3 ubiquitin‐protein ligase listerin (LTN1), Ribosome quality control complex subunit NEMF (NEMF), and E3 ubiquitin‐protein ligase RNF10 (RNF10) in various KO HEK293T cells. (B) Proteomics‐based expression profile of LTN1 across various mouse tissues . Dark blue indicates a higher expression level. This figure was prepared by the authors of this article. (C) Western blot validation of LTN1, NEMF and RNF10 protein levels in mouse tissues. Ponceau stain was used as a loading control. (D) Western blot analysis of RNF10 expression in the mouse pancreatic β‐cell line MIN6 expressing sgRNA. RNF10 expression was strongly increased in LTN1‐deficient cells (lanes 1 and 3). (E) The uS3 ubiquitination level was detected by western blotting using anti‐uS3 antibody. Mono‐ubiquitinated uS3 was indicated by the arrow.

Article Snippet: DNA was amplified from cDNA of HEK293T or MIN6 cells using KOD FX Neo polymerase (KFX‐201; TOYOBO, Kita, Osaka, Japan) and cloned into the vector.

Techniques: Western Blot, Ubiquitin Proteomics, Control, Expressing, Biomarker Discovery, Staining

Journal: Febs Letters

Article Title: Crosstalk between the ribosome quality control‐associated E3 ubiquitin ligases LTN1 and RNF10

doi: 10.1002/1873-3468.70230

Figure Lengend Snippet: The increase in RNF10 protein levels is partially dependent on upregulation of mRNA. Relative expression levels of RNF10 mRNA and protein were quantified by RT‐qPCR and western blotting. Vertical axes indicate relative expression level of mRNA/protein normalized by GAPDH compared to WT. Error bars represent mean ± SEM of three independent experiments. Statistical significance was evaluated using a two‐tailed unpaired Student's t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001. N.S. not significant. (A)HEK293T (B) MIN6.

Article Snippet: DNA was amplified from cDNA of HEK293T or MIN6 cells using KOD FX Neo polymerase (KFX‐201; TOYOBO, Kita, Osaka, Japan) and cloned into the vector.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Febs Letters

Article Title: Crosstalk between the ribosome quality control‐associated E3 ubiquitin ligases LTN1 and RNF10

doi: 10.1002/1873-3468.70230

Figure Lengend Snippet: Loss of LTN1 reduces ribosome pausing on RNF10 mRNA. (A) Schematic diagram of the ribosome‐profiling workflow. (B) Ribosome‐footprint tracks on the RNF10 mRNA in MIN6 cells expressing sgNT (gray) or sgLTN1(red). Position of P‐site was displayed. (C) Diagram of the flow‐cytometry reporter; the full‐length mouse RNF10 coding sequence was inserted into the gray segment (X region). (D) Representative flow‐cytometry profiles of reporters without an insert (no insert) and with the full‐length RNF10 insert in sgNT (blue) and sgLTN1 (red) expressing MIN6 cells. (E) Bar graph of the relative median mCherry/GFP fluorescence ratio of the RNF10 reporter shown in (D). Error bars represent mean ± SEM of 8 independent experiments. Statistical significance was evaluated using a two‐tailed unpaired Student's t ‐test. * P < 0.05.

Article Snippet: DNA was amplified from cDNA of HEK293T or MIN6 cells using KOD FX Neo polymerase (KFX‐201; TOYOBO, Kita, Osaka, Japan) and cloned into the vector.

Techniques: Expressing, Flow Cytometry, Sequencing, Fluorescence, Two Tailed Test

Journal: Febs Letters

Article Title: Crosstalk between the ribosome quality control‐associated E3 ubiquitin ligases LTN1 and RNF10

doi: 10.1002/1873-3468.70230

Figure Lengend Snippet: LTN1 regulates RNF10 expression level via its RING domain. (A) The GFP‐K20‐HIS3 reporter and LTN1 constructs were co‐expressed in HEK293T cells. Both the full‐length product and the arrest product were detected. (B, D) Western blot analysis of RNF10 protein levels in LTN1‐knockout HEK293T cells (B) and sgLTN1 MIN6 cells (D) co‐expressing either wild‐type LTN1 or the RING domain deletion mutant (ΔRING). (C, E) Quantified value of RNF10 expression levels shown in (B) and (D), respectively. RNF10 levels were normalized to GAPDH and expressed relative to those in WT or sgNT cells transfected with an empty vector (EV). Data represent mean ± SEM from three independent experiments. Statistical significance was assessed using a two‐tailed unpaired Student's t ‐test. * P < 0.05; N.S., not significant.

Article Snippet: DNA was amplified from cDNA of HEK293T or MIN6 cells using KOD FX Neo polymerase (KFX‐201; TOYOBO, Kita, Osaka, Japan) and cloned into the vector.

Techniques: Expressing, Construct, Western Blot, Knock-Out, Mutagenesis, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Febs Letters

Article Title: Crosstalk between the ribosome quality control‐associated E3 ubiquitin ligases LTN1 and RNF10

doi: 10.1002/1873-3468.70230

Figure Lengend Snippet: Effects of LTN1 depletion on translation. (A) MA plot showing differences in ribosome load between sgLTN1 and sgNT MIN6 cells. Genes with baseMean > 10, adjusted P ‐value (padj) < 0.1, and absolute log 2 fold change > 0.2 are highlighted in red (upregulated) or blue (downregulated). (B) Results of KEGG pathway over‐representation analysis of upregulated genes. (C) Western blot analysis of RNF10 protein levels in ubiquitin‐fold modifier 1 (UFM1) or Ufm1‐specific protease 2 (UFSP2) KO HEK293T cells. (D) Quantified value of RNF10 expression levels shown in (C). RNF10 levels were normalized to GAPDH and expressed relative to those in WT cells. Data represent mean ± SEM from three independent experiments. Statistical significance was assessed using a two‐tailed unpaired Student's t ‐test. * P < 0.05, ** P < 0.01. (E) Proposed model. Knockout of LTN1, UFM1, or UFSP2 impairs ER‐associated ribosome quality control (ER‐RQC), which may activate compensatory mechanisms. Upregulation of RNF10, together with increased uS3 mono‐ubiquitination, could represent an adaptive response to ER‐RQC defects, helping to maintain cellular homeostasis. LTN1 regulates the expression level of RNF10 via its RING domain.

Article Snippet: DNA was amplified from cDNA of HEK293T or MIN6 cells using KOD FX Neo polymerase (KFX‐201; TOYOBO, Kita, Osaka, Japan) and cloned into the vector.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Two Tailed Test, Knock-Out, 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‐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: 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