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: 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

Journal: Science Advances

Article Title: 3D bioprinting of collagen-based high-resolution internally perfusable scaffolds for engineering fully biologic tissue systems

doi: 10.1126/sciadv.adu5905

Figure Lengend Snippet: ( A ) Schematic design and 3D printer machine pathing G-code of a dual parallel channel multi-material CHIPS with pancreatic vascular cell bioink (MIN6, HUVEC, and MSC) regions surrounding both sides of the channels. ( B ) Pancreatic CHIPS FRESH printed and visualized via bright-field stereomicroscope (inset) and 3D confocal fluorescence imaging of the optically cleared pancreatic scaffold after 12 days of static culture. ( C ) XY midplane view from confocal fluorescence image of the 12-day statically cultured pancreatic CHIPS following 3D vascular network segmentation for quantification of network diameter and density within the migratory zones. ( D ) Example confocal fluorescence images revealing additional cell migration into the acellular regions of the CHIPS beneath the cellular regions guided by the printed collagen filaments. ( E ) XY midplane projection view from 3D confocal fluorescence imaging of the optically cleared 12-day VAPOR perfused pancreatic CHIPS. ( F ) Graphic illustration and example ROIs depicting evidence of early MIN6 pancreatic bud and microlumen formation with actin (green) and insulin (magenta) fluorescence images from ROIs 2 and 3 in (E). ( G ) Graphic illustration and quantification for insulin secretion ELISA assay from 1.5-hour glucose-stimulated [ratio of high glucose (HG) to low glucose (LG)] insulin secretion experiment between 12-day static and perfusion cultured pancreatic CHIPS (means ± SD; ** P < 0.01 for N = 3 static tissues; N = 2 perfused tissues, unpaired t test).

Article Snippet: MIN6 (mouse insulinoma cell line) (Addexbio, C0018008) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 11-965-092) with 15% fetal bovine serum (v/v), 2 mM sodium pyruvate, 20 mM Hepes, and 0.05 mM β-mercaptoethanol, using passages 9 to 15.

Techniques: Fluorescence, Imaging, Cell Culture, Migration, Enzyme-linked Immunosorbent Assay