Journal: Scientific Reports

Article Title: Plasmonic imaging of living pancreatic beta-cell networks

doi: 10.1038/s41598-025-34094-0

Figure Lengend Snippet: Surface Plasmon Resonance Microscopy (SPRM) of pancreatic beta-cells ( a ) Schematic presenting the experimental setup for SPRM, depicting the layered structure of gold thin film on glass substrate with cells adhered to the gold surface in Hanks’ balanced salt solution (HBSS). A fiber-coupled laser (690 nm) is collimated before focusing on the back focal plane (BFP) of a high numerical aperture oil immersion objective to produce a collimated beam at the sample. The angle of illumination is varied by laterally scanning the focus on the BFP. The sample is imaged using a 2D CMOS pixelated detector. ( b ) A magnified view of the SPR sensor and cell interface showing the interface layers (glass, Au thin film of 50 nm, medium (HBSS), cell membrane of c. 7 nm thickness, and cytosol), with an illustration of the penetration depth of SPs in both metal and dielectric media. This indicates sensitivity to the cell membrane and the proximal intra- and extracellular spaces. ( c ) SPR curves presenting the reflection coefficient for various angles of incidence, simulated for bare gold with HBSS and for the gold-cell interface respectively. ( d ) The corresponding first derivative of reflectivity with respect to the angle of incidence, showing the variations in the sensitivity of the measurement for optimising the angle of illumination. ( e ) (i). Brightfield microscopy image of live MIN6 beta-cells cultured on PLL-modified Au thin film. e (ii), e (iii), and e (iv) are the corresponding SPRM images at different angles of illumination. The angle of incidence is selected in region iii, although this gives reduced sensitivity, it allows simultaneous tracking of cells and the extracellular regions where cells are not present on the sensor.

Article Snippet: The mouse pancreatic cell line, MIN6 (Beta-TC-6, ATCC; CRL-11506), was maintained in high Glucose DMEM (Merck, D5671) supplemented with 10% FBS (Merck, F9665), 10 mM HEPES (Merck, R0887), 50 mg/ml penicillin and streptomycin (Merck, P0781) and 50 mM b-mercaptoethanol (Merck, M3148).

Techniques: SPR Assay, Microscopy, Membrane, Cell Culture, Modification

Journal: Scientific Reports

Article Title: Plasmonic imaging of living pancreatic beta-cell networks

doi: 10.1038/s41598-025-34094-0

Figure Lengend Snippet: SPRM reveals correlated oscillations in pancreatic beta-cells. ( a ) Brightfield image of MIN6 cells cultured on PLL-modified Au thin film. ( b ) Corresponding SPRM image with five regions of interest highlighting cells (1, 2 and 3) and the extracellular background (4 and 5). ( c ) Time-resolved reflectivity recorded over 130 s in HBSS with 10 mM glucose for the five regions shown in ( b ), inset shows a magnified view of a selected time window, indicated by t to t’, for the three cells which shows synchronised intensity oscillations. Traces appear synchronised but the background ROIs are anticorrelated. ( d ) Heat map displaying the correlation between signals extracted from ROIs 1 – 5 investigating signaling at the cellular ROIs (1–3) and the background ROIs (4, 5), where cells are not present.

Article Snippet: The mouse pancreatic cell line, MIN6 (Beta-TC-6, ATCC; CRL-11506), was maintained in high Glucose DMEM (Merck, D5671) supplemented with 10% FBS (Merck, F9665), 10 mM HEPES (Merck, R0887), 50 mg/ml penicillin and streptomycin (Merck, P0781) and 50 mM b-mercaptoethanol (Merck, M3148).

Techniques: Cell Culture, Modification

Journal: Scientific Reports

Article Title: Plasmonic imaging of living pancreatic beta-cell networks

doi: 10.1038/s41598-025-34094-0

Figure Lengend Snippet: Intracellular calcium oscillations in pancreatic beta cells within HBSS supplemented with 10 mM glucose. ( a ) Fluorescence image of MIN6 cells loaded with FLUO-4, excited at 450–490 nm, with 0–50 regions of interest (ROIs) indicated (0 denotes the background). Average fluorescence image of 3,000 frames of a field of MIN6 cells, with each measured cell labelled to indicate its 2D geography . ( b ) Cross-correlation matrix showing the Pearson correlation coefficients (threshold of 0.3) calculated between ROIs 0–50. ( c ) Exemplary 100-s traces of selected ROIs (band-pass filtered between 0.1 and 5 Hz), including clusters with high correlation {C8, C9}, {C11– C15}, {C16–C21}, {C23, C24}, {C44, C45}, alongside other ROIs selected at random.

Article Snippet: The mouse pancreatic cell line, MIN6 (Beta-TC-6, ATCC; CRL-11506), was maintained in high Glucose DMEM (Merck, D5671) supplemented with 10% FBS (Merck, F9665), 10 mM HEPES (Merck, R0887), 50 mg/ml penicillin and streptomycin (Merck, P0781) and 50 mM b-mercaptoethanol (Merck, M3148).

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Plasmonic imaging of living pancreatic beta-cell networks

doi: 10.1038/s41598-025-34094-0

Figure Lengend Snippet: Synchronised network oscillations are suppressed in the presence of a calcium channel blocker. ( a ) Brightfield image of MIN6 cells cultured on PLL-modified Au thin film. Scale bar 10 μm. ( b ) Corresponding SPRM image with 6 cellular regions of interest. ( c ) Time-series recordings from the six cellular ROIs presenting time-resolved reflectivity, under treatment with: 1) Hanks balanced salt solution (HBSS) without glucose; 2) HBSS supplemented with 10 mM glucose; and 3) HBSS supplemented with 10 mM glucose and 40 µM nifedipine. ( d ) Comparison of the effect of the three treatments on cells displaying the average amplitude profiles of the cells. Prior to identifying the amplitude profile, each signal was filtered between 0.1–15 Hz (see Methods) before standardization using the standard deviation over all the three recordings. Pairwise comparisons were performed using paired t‑tests, with p‑values adjusted for multiple comparisons (n = 6 cells) using the Bonferroni correction. Whiskers extend to 1.5 times the interquartile range (IQR).

Article Snippet: The mouse pancreatic cell line, MIN6 (Beta-TC-6, ATCC; CRL-11506), was maintained in high Glucose DMEM (Merck, D5671) supplemented with 10% FBS (Merck, F9665), 10 mM HEPES (Merck, R0887), 50 mg/ml penicillin and streptomycin (Merck, P0781) and 50 mM b-mercaptoethanol (Merck, M3148).

Techniques: Cell Culture, Modification, Comparison, Standard Deviation

Journal: Scientific Reports

Article Title: Plasmonic imaging of living pancreatic beta-cell networks

doi: 10.1038/s41598-025-34094-0

Figure Lengend Snippet: Glucose modulation of MIN6 electrical behaviour assessed using MEA recordings. ( a ) Bright-field micrograph of the circular microelectrode array used for recordings, displaying the radial arrangement of electrodes and the central culture region where MIN6 cells were seeded. Scale bar = 1000 μm. ( b ) Impedance-based viability maps for three independent wells (W1-W3). Each heatmap shows the impedance magnitude measured at the electrode–cell interface, serving as a surrogate metric for cell coverage and viability. Higher impedance indicates greater cell attachment. The four rows depict: no glucose, 10 mM glucose, 2 μM nifedipine, and two days after nifedipine treatment, illustrating condition-dependent variations in cell viability and adherence. ( c ) Representative 100-s extracellular voltage traces recorded from the same MEA electrode under three conditions: glucose-free HBSS, HBSS supplemented with 10 mM glucose, and HBSS supplemented with 10 mM glucose plus 2 μM nifedipine. The traces, filtered with a standard 0–15 Hz band-pass, reveal condition-dependent variations in the amplitude of MIN6 electrical activity. ( d ) Frequency-resolved decomposition of the same electrode shown in ( a ). Glucose enhances electrical activity, while nifedipine suppresses it within the 1–15 Hz range.

Article Snippet: The mouse pancreatic cell line, MIN6 (Beta-TC-6, ATCC; CRL-11506), was maintained in high Glucose DMEM (Merck, D5671) supplemented with 10% FBS (Merck, F9665), 10 mM HEPES (Merck, R0887), 50 mg/ml penicillin and streptomycin (Merck, P0781) and 50 mM b-mercaptoethanol (Merck, M3148).

Techniques: Microelectrode Array, Cell Attachment Assay, Activity Assay

Journal: Scientific Reports

Article Title: Plasmonic imaging of living pancreatic beta-cell networks

doi: 10.1038/s41598-025-34094-0

Figure Lengend Snippet: Cell-attached patch-clamp recordings of glucose-induced activity in a MIN6 β-cell and quantification of spike frequency. ( a ) Representative current trace recorded under three consecutive conditions: HBSS buffer (0 mM glucose), 10 mM glucose, and 10 mM glucose + 10 µM nifedipine. The black trace shows the analysed current, while grey segments correspond to periods of perfusion during which mechanical noise was introduced. Red ticks mark automatically detected downward current deflections identified as action-current events using a dynamic threshold-based detection algorithm (threshold = baseline – 3 × noise; 50 ms refractory period). Coloured horizontal bars indicate the duration of each condition (blue = HBSS, yellow = HBSS + glucose, green = HBSS + glucose + nifedipine). Expanded regions below illustrate zoomed view of spike events during the baseline and glucose phases, with an inset showing a single representative event (amplitude ≈ 3 pA, width ≈ 0.02 ms). ( b ) Quantification of firing activity for the three conditions for three experiments. Each box represents the distribution of windowed spike rates (10-s windows, 2-s step) across three cells analysed (286 spike events). Box edges denote the inter-quartile range (25th–75th percentile); the centre line shows the median; whiskers extend to 1.5 times the IQR. Diamond symbols indicate the mean rate for each condition. Mean ± SEM firing rates were 0.88 ± 0.06 spikes/s for 0 mM glucose, 1.57 ± 0.07 spikes/s for 10 mM glucose, and 0.26 ± 0.02 spikes/s for 10 mM glucose + 10 µM nifedipine. Nifedipine effectively suppressed activity, consistent with its role as a channel blocker.

Article Snippet: The mouse pancreatic cell line, MIN6 (Beta-TC-6, ATCC; CRL-11506), was maintained in high Glucose DMEM (Merck, D5671) supplemented with 10% FBS (Merck, F9665), 10 mM HEPES (Merck, R0887), 50 mg/ml penicillin and streptomycin (Merck, P0781) and 50 mM b-mercaptoethanol (Merck, M3148).

Techniques: Patch Clamp, Activity Assay

Journal: Scientific Reports

Article Title: Plasmonic imaging of living pancreatic beta-cell networks

doi: 10.1038/s41598-025-34094-0

Figure Lengend Snippet: Network analysis. ( a, b ) Brightfield and SPRM images of MIN6 cells, respectively. Scale bar 10 μm. ( c ) Connectivity matrices for the following conditions: baseline HBSS ( c.i ), HBSS supplemented with 10 mM glucose ( c.ii ) HBSS supplemented with 10 mM glucose and 40 μM nifedipine ( c.iii ). ( d ) Corresponding directed graphs represent cells ROIs as nodes, with edges (i.e. arrows) indicating patterns of directional connectivity and their associated weights. ( e ) Panels e(i) to e(iii) show examples of time series and their associated amplitude envelopes. Time-resolved connectivity, is presented for each of the above experimental conditions, measured via phase locking factor (PLF) and compared to amplitude correlation coefficient (ACC). PLF was calculated for a 10-s window with one second overlaps, for all cells and for each treatment. Similarly, ACC was computed by obtaining undirected correlation between the amplitude envelopes. ( f ) Boxplots depicting the mean undirected PLF (i) and the mean ACC (ii), calculated from their respective dynamic observations and averaged across six cells, are presented in panels e(i) and e(ii). In each boxplot, horizontal lines indicate the median values, while the boxes represent the IQR. Whiskers extend to 1.5 times the IQR. Paired t-tests were performed for multiple comparisons, with all P-values adjusted using the Bonferroni correction.

Article Snippet: The mouse pancreatic cell line, MIN6 (Beta-TC-6, ATCC; CRL-11506), was maintained in high Glucose DMEM (Merck, D5671) supplemented with 10% FBS (Merck, F9665), 10 mM HEPES (Merck, R0887), 50 mg/ml penicillin and streptomycin (Merck, P0781) and 50 mM b-mercaptoethanol (Merck, M3148).

Techniques:

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