Journal: PLoS ONE

Article Title: Lack of Wdr13 Gene in Mice Leads to Enhanced Pancreatic Beta Cell Proliferation, Hyperinsulinemia and Mild Obesity

doi: 10.1371/journal.pone.0038685

Figure Lengend Snippet: A) Transfection with Ad Wdr 13 and AdGFP viruses shows overexpression of WDR13 protein in MIN6 cells as visualized by immunoblotting using anti WDR13 antibody. Lower panel shows beta actin as loading control. Overexpression of WDR13 protein results in retardation in cell proliferation after 48 h of transfection with 100 MOI. B) Overexpression of WDR13 protein results in accumulation of p21, whereas Cyclin D1, Cyclin D2, Cyclin E1 and p27 levels remain unaffected. C) siRNA knockdown of WDR13 in MIN6 cells. MIN6 cells were transfected with nonspecific scrambled (Scr) siRNAs and WDR13 specific siRNA. Immunoblot analysis shows Knockdown of WDR13 protein. Actin was used as loading control. Immunoblot, using p21 antibody shows reduction of p21 levels in WDR13 knockdown MIN6 cell. D) Cell cycle inhibitor p21 expression in purified pancreatic islets of Wdr 13 knockout mice and that of wild type littermates by western blot analysis. Beta actin was used as loading control. p21 expression is less in the islets of knockout mice. E) Occupancy by WDR13 at p21 promoter revealed by chromatin immunoprecipitation using primers specific for p21 and GAPDH.

Article Snippet: 10,000 MIN6 cells (obtained from National Centre for Cell Science, Pune, India) were seeded per well of 24 well plates in DMEM media containing 10% FBS and were transfected either with AdGFP or Ad Wdr13 using a titer of 100 MOI.

Techniques: Transfection, Over Expression, Western Blot, Control, Knockdown, Expressing, Purification, Knock-Out, Chromatin Immunoprecipitation

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Images of the spheroids from static culture for morphological analysis and the determination of properties such as spheroid size and circularity ( n = 12; data are means ± STDV). (A) Morphological differences can be seen during the aggregation of the different β‐cell lines within 7 days. The images of INS‐1, MIN6, and MSCs were reduced by 60%, whereas the 1.1B4 images were reduced by 73% ( * ). In both cases the scale bar represents 100 μm. (B) The growth kinetics of the spheroids reveal the differences between cell lines. The INS‐1 cells (squares) showed fast aggregation and a continuous increase in spheroid size, whereas the MIN6 cells (circles) needed 3 days to form stable spheroids before slow growth was observed. The 1.1B4 cells (triangles) aggregated slowly over 5–7 days, represented by a peak on day 2 followed by a decline in size until a stable spheroid was formed and a volume increase was observed. The hMSC‐TERTs (crosses) and ad‐MSCs (stars) showed no growth and the spheroid size declined over time. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell; STDV, standard deviations

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: The viability of spheroids from static cultures determined by staining with calcein AM and ethidium after 7 days. (A) INS‐1 spheroids featured a dead core and a viable mantle, whereas MIN6 spheroids featured a heterogenous distribution of dead cells. The loose structure of the 1.1B4 spheroids promoted sufficient mass transfer resulting in a high viability. (B) Insulin secretion profiles of INS‐1 cells cultured as monolayers and spheroids cultured under static (96‐well plate) and dynamic (shaking flask) conditions ( n = 3; data are means ± STDV; significance intervals are * p < 0.05, ** p < 0.01, and *** p < 0.001). 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; STDV, standard deviations

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Staining, Cell Culture, Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: The insulin profiles of β‐cell spheroids in static culture was measured by GSIS ( n = 3; error = STDV)

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques:

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Aggregation of INS‐1 (upper row), MIN6 (middle row) and 1.1B4 (lower row) cells with hMSC‐TERTs at different ratios after 24 h. MSCs were stained blue (VPD) and β‐cells were stained with the green dye CFSE. Starting with monospheroids in the first (MSCs, blue) and second (β‐cells, green) columns, the cell ratios increase from left to right. Due to different scaling of the images, the MSC spheroids seem to have a different size in each setup, but the seeding density was always 1000 cells per well. In all cases the scale bar represents 100 μm. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; CFSE, 5‐(and 6)‐carboxyfluorescein diacetate, succinimidyl ester; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Staining, Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Bright‐field and viability images (at day 7) of monospheroids (0–1 = MSC only; 1–0 = β‐cell only) and heterospheroids INS‐1 (upper row), MIN6 (middle row), and 1.1B4 (lower row) cocultured with hMSC‐TERTs at different cell ratios. The stated viabilities of the spheroids were assessed by measuring the red (core) and green (whole spheroid) diameter and the resulting volume to describe the real “3D viability,” but the displayed images only represent two dimensions of the spheroids, which could provide a deceptive impression. Scale bar = 100 μm. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Electrofusion

Journal: Nature Communications

Article Title: THADA inhibition in mice protects against type 2 diabetes mellitus by improving pancreatic β-cell function and preserving β-cell mass

doi: 10.1038/s41467-023-36680-0

Figure Lengend Snippet: a Representative pancreatic sections from non-diabetes (ND) and type 2 diabetes mellitus (T2DM) participants stained for insulin (green), THADA (red), and DAPI (blue). Scale bars, 50 μm. b THADA intensity of each islet in ND and T2DM participants ( n = 10 for #29 and #74, n = 12 for #96, n = 13 for #30, n = 14 for #01 and #11, n = 15 for #07, n = 16 for #89). c Quantification of mean THADA intensities in ND and T2DM individuals ( n = 4). d Representative pancreatic sections from 16-week-old db/db mice and their wild-type control littermates stained for insulin (green), THADA (red), and DAPI (blue). Scale bars, 50 μm. e Quantification of THADA intensities in control littermates and db/db mice at 4, 8, and 16 weeks of age ( n = 3). f Linear regression analysis of islet THADA intensities and blood glucose levels in control littermates (circles) and db/db mice (triangles). The R 2 and P value were shown in panel. g Representative pancreatic sections from mice feeding HFHS diet for 12 weeks or normal chow diet (NCD) staining for insulin (green), THADA (red), and DAPI (blue). Scale bars, 50 μm. h Quantification of mean THADA intensities in NCD- and HFHS diet-fed mice ( n = 3). i Thada mRNA expression in mouse islets incubated with 3.3 mM glucose (LG), 0.5 mM palmitate (PA), 16.7 mM glucose (HG), or 16.7 mM glucose combined with 0.5 mM palmitate (HGPA) for 48 h ( n = 3 biologically independent experiments). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; significance is assessed by two-tailed unpaired Student’s t test ( c , e ) or one-way ANOVA followed by Dunnett’s multiple comparison test ( i ). Source data are provided as a Source Data file.

Article Snippet: Stable MIN6 beta-cell line with endogenous Thada -activation was generated by CRISPR/dCas9-SAM system from Genechem (Shanghai, China).

Techniques: Staining, Control, Expressing, Incubation, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: THADA inhibition in mice protects against type 2 diabetes mellitus by improving pancreatic β-cell function and preserving β-cell mass

doi: 10.1038/s41467-023-36680-0

Figure Lengend Snippet: a Body weights of wild-type (WT, n = 13), heterozygous (HET, n = 13), and Thada -knockout (KO, n = 11) mice at the indicated weeks of age. b Fasting blood glucose levels of WT ( n = 10), HET ( n = 8) and Thada -KO mice ( n = 5) that were fasted overnight. c Random-fed blood glucose levels of WT ( n = 11), HET ( n = 13), and Thada -KO mice ( n = 10). d Fasting ( n = 8) and fed ( n = 8 for WT and n = 5 for KO) serum insulin levels of WT and Thada -KO mice. e , f IPGTT and the related area under the curve (AUC) of WT ( n = 9), HET ( n = 6) and Thada -KO mice ( n = 7). g , h ITT and the related area under baseline (AUB) of WT and Thada -KO mice ( n = 6). ( i ) Body weights of control ( Thada fl/fl ) and β-cell-specific Thada -knockout (β Thada -KO) mice ( n = 5). j , k 16-h fasting blood glucose ( j ) and random-fed blood glucose ( k ) levels of Thada fl/fl and β Thada -KO mice ( n = 5). l Fed serum insulin levels of Thada fl/fl ( n = 7) and β Thada -KO ( n = 5) mice. m , n IPGTT and the related AUC of Thada fl/fl and β Thada -KO mice ( n = 5). o , p ITT and the related AUB of Thada fl/fl and β Thada -KO male mice ( n = 5). Experiments were performed on male mice at 8–10 weeks of age and female mice data were provided in Fig. . Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01; significance is assessed by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Article Snippet: Stable MIN6 beta-cell line with endogenous Thada -activation was generated by CRISPR/dCas9-SAM system from Genechem (Shanghai, China).

Techniques: Knock-Out, Control, Two Tailed Test

Journal: Nature Communications

Article Title: THADA inhibition in mice protects against type 2 diabetes mellitus by improving pancreatic β-cell function and preserving β-cell mass

doi: 10.1038/s41467-023-36680-0

Figure Lengend Snippet: a Serum insulin levels at 0, 15, and 30 min after intraperitoneal glucose injection in WT ( n = 13) and Thada -KO mice ( n = 8). b Area under the curve in ( a ). c Pancreatic insulin contents of WT ( n = 5) and Thada -KO mice ( n = 3). d Islets isolated from WT and Thada -KO mice were stimulated with 3.3, 11.1, or 16.7 mM glucose for 1 h and insulin secretion was assayed. Stimulation index was calculated as fold change of GSIS ( n = 5). e Islets isolated from WT and Thada -KO mice were stimulated with or without 35 mM KCl at 3.3 mM glucose for 1 h and insulin secretion was assayed ( n = 3). f ATP content of islets from WT and Thada -KO mice incubated at 3.3 or 16.7 mM glucose for 1 h ( n = 3). g Transmission electron microscopy of pancreatic islets from WT and Thada -KO mice. Red and blue arrowheads point to vesicles containing immature and mature granules, respectively. Scale bar, 2 μm. h – j Analysis of ultrastructural β-cells from WT and Thada -KO mice ( n = 3), including quantification of the percentage of mature and immature vesicles ( h ), vesicle density ( i ), and vesicle diameter ( j ). k MIN6 beta-cell lines were transduced with dCas9-SAM lentiviruses to activate Thada expression, then GSIS was assayed at 1.4 mM, 5.6 mM, and 25 mM glucose ( n = 5 for control cells and n = 6 for Thada -SAM cells). l Control (CON, n = 3) and Thada -activated ( Thada -SAM, n = 4) MIN6 beta-cell lines were stimulated at 2.8 mM glucose with or without 2.5 mM tolbutamide and 35 mM KCl for 1 h and insulin secretion was assayed. ( m ) Islets transduced with dCas9-SAM lentiviruses for 7 days were then stimulated with 3.3 and 16.7 mM glucose for 1 h and insulin secretion was assayed ( n = 4). n Transmission electron microscopy of control and Thada -SAM MIN6 beta-cell lines. Red and blue arrowheads point to vesicles containing immature and mature granules, respectively. Scale bar, 2 μm. o – q Quantification of the percentage of mature and immature vesicles ( o ), vesicle density ( p ), and vesicle diameter ( q ) in control and Thada -SAM MIN6 beta-cell lines ( n = 3). The animal experiments were performed on male mice at 8–10 weeks of age and female mice data were provided in Fig. . Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; significance is assessed by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Article Snippet: Stable MIN6 beta-cell line with endogenous Thada -activation was generated by CRISPR/dCas9-SAM system from Genechem (Shanghai, China).

Techniques: Injection, Isolation, Incubation, Transmission Assay, Electron Microscopy, Transduction, Expressing, Control, Two Tailed Test

Journal: Nature Communications

Article Title: THADA inhibition in mice protects against type 2 diabetes mellitus by improving pancreatic β-cell function and preserving β-cell mass

doi: 10.1038/s41467-023-36680-0

Figure Lengend Snippet: a Representative immunohistochemistry images of pancreatic sections from WT and Thada -KO mice stained for insulin (brown) and eosin (red). Scale bars, 1 mm for whole pancreas and 200 μm for magnified image. b Measurements of β-cell area/pancreatic area ratio in WT and Thada -KO mice ( n = 4). c Measurements of β-cell mass in WT and Thada -KO mice ( n = 4). d Representative immunofluorescence images of islets from WT and Thada -KO mice stained for insulin (green), Ki67 (red), and DAPI (blue). Scale bars, 20 μm. Arrowhead points to Ki67 + Insulin + cells. e The proliferation of β-cell was determined by quantification the percentage of Ki67 + in Insulin + cells ( n = 4 mice each group). 3428 ± 600 Insulin + cells were quantified for WT mice and 3328 ± 533 Insulin + cells were quantified for KO mice. f Representative immunofluorescence images of islets from WT and Thada -KO mice stained for insulin (green), Tunel (red), and DAPI (blue). Scale bars, 20 μm. Arrowhead points to Tunel + Insulin + cells. g The apoptosis of β-cell was determined by quantification the percentage of Tunel + in Insulin + cells ( n = 4 mice each group). 4204 ± 871 Insulin + cells were quantified for WT mice and 4568 ± 591 Insulin + cells were quantified for KO mice. h Flow cytometry analysis of control and Thada -SAM MIN6 beta-cell lines treated with or without 1 μM thapsigargin (Tg) or 1 μg/ml tunicamycin (Tm) for 24 h. Representative dot plots of cell apoptosis were shown after dual staining with Annexin V and 7-AAD. The gating strategy was provided in Fig. . i The apoptosis of control and Thada -SAM MIN6 beta-cell lines were quantified by the percentage of Annexin V-positive cells ( n = 4). The animal experiments were performed on male mice at 8-10 weeks of age and female mice data were provided in Figure . Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01; significance is assessed by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Article Snippet: Stable MIN6 beta-cell line with endogenous Thada -activation was generated by CRISPR/dCas9-SAM system from Genechem (Shanghai, China).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, TUNEL Assay, Flow Cytometry, Control, Two Tailed Test

Journal: Nature Communications

Article Title: THADA inhibition in mice protects against type 2 diabetes mellitus by improving pancreatic β-cell function and preserving β-cell mass

doi: 10.1038/s41467-023-36680-0

Figure Lengend Snippet: a Increases in [Ca 2+ ] i from control (CON) and Thada -activated ( Thada -SAM) MIN6 beta-cell lines following stimulation with 2.8 mM glucose (2.8 G), 25 mM glucose (25 G) and 30 mM KCl. Representative results of three replicates from each group are provided. b Quantification of the maximum increases in [Ca 2+ ] i after 25 mM glucose ( n = 13 for control and n = 11 for Thada -SAM) or 30 mM KCl stimulation. ( n = 14 for control and n = 11 for Thada -SAM). c , d Quantification of the maximum increases in [Ca 2+ ] i from control ( n = 6) and Thada -SAM β-cells ( n = 5) after 10 μM ionomycin stimulation. Representative results of two replicates from each group are provided. e , f Quantification of the maximum increases in [Ca 2+ ] i from control ( n = 6) and Thada -SAM β-cells ( n = 7) after 2 μM thapsigargin stimulation. Representative results of two replicates from each group are provided. g [Ca 2+ ] i in dispersed islet cells from WT and Thada -KO mice at basal 2.8 mM glucose and after stimulation with 16.7 mM glucose (16.7 G) ( n = 16 from six mice each group). h Quantification of the maximum increases in [Ca 2+ ] i after 16.7 mM glucose stimulation ( n = 16 from six mice each group). i , j Quantification of [Ca 2+ ] i in dispersed islet cells from WT and Thada -KO mice after stimulation with 35 mM KCl at 2.8 mM glucose ( n = 16 from six mice each group). k , l Quantification of [Ca 2+ ] i in dispersed islet cells from WT and Thada -KO mice after 10 μM ionomycin stimulation ( n = 16 from six mice each group). m Immunofluorescence staining for THADA (green), SERCA2 (red), and DAPI (blue) in MIN6 beta-cell line. Scale bars, 10 μm. n Immunofluorescence staining for THADA (green), RyR (red), and DAPI (blue) in MIN6 beta-cell line. Scale bars, 10 μm. o MIN6 cells were immunoprecipitated with either a SERCA2 antibody or an IgG negative control, followed by western blot analysis using THADA and SERCA2 antibodies. p MIN6 cells were immunoprecipitated with either a RyR antibody or an IgG negative control, followed by western blot analysis using THADA and RyR antibodies. q Proximity ligation assay for THADA together with SERCA2 and RyR in MIN6 beta-cell line (NC for negative control). Scale bars, 10 μm. r Increases in [Ca 2+ ] i from control and Thada -activated β-cells pretreated with or without 20 μM S107 or 10 μM CDN1163 (CDN) after 10 μM ionomycin stimulation. Representative results from each group are presented ( n = 10 for control, n = 12 for Thada -SAM, n = 11 for Thada -SAM with S107 and CDN1163). s Control and Thada -SAM β-cells were pretreated with or without CDN1163 and S107 at the indicated concentration for 24 h, then stimulated with 1.4 mM glucose (1.4 G) or 25 mM glucose for 1 h, and insulin secretion was assayed ( n = 4, except for control cells at 25 G and Thada -SAM cells treated with 10 μM S107 at 25 G were n = 3). All western blots and immunostainings (m-q) show representative results from at least three independent experiments. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; significance is assessed by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Article Snippet: Stable MIN6 beta-cell line with endogenous Thada -activation was generated by CRISPR/dCas9-SAM system from Genechem (Shanghai, China).

Techniques: Control, Immunofluorescence, Staining, Immunoprecipitation, Negative Control, Western Blot, Proximity Ligation Assay, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: THADA inhibition in mice protects against type 2 diabetes mellitus by improving pancreatic β-cell function and preserving β-cell mass

doi: 10.1038/s41467-023-36680-0

Figure Lengend Snippet: a The caspase-3/7 activities of control and Thada -SAM β-cells were assayed after treatment with 1 μM thapsigargin (Tg) in the presence or absence of 10 μM CDN1163 for 24 h ( n = 6). b Control and Thada -SAM β-cells were treated with or without 0.5 mM palmitate (Palm), 1 μM thapsigargin or 1 μg/ml tunicamycin (Tm) for 24 h, then cleaved caspase-8, cleaved caspase-3, CHOP and ATF4 protein levels were determined. c The caspase-8 activities of control ( n = 6) and Thada -SAM ( n = 8) β-cells were assayed after treatment with or without 1 μM Tg for 24 h. d The caspase-3/7 activities of control and Thada -SAM β-cells were assayed after treatment with vehicle ( n = 5 for control and n = 3 for Thada -SAM) or 1 μM Tg ( n = 6) for 24 h. e , f Control and Thada -KO MIN6 beta-cell lines were treated with 1 μM Tg ( e ) or 1 μg/ml Tm ( f ) for 24 h, then cleaved caspase-8 and cleaved caspase-3 protein levels were determined. g MIN6 beta-cell lines were treated with 1 μM Tg for 24 h, then immunoprecipitated with either an IgG control or a FADD antibody, followed by western blot analysis with a THADA antibody. h Control and Thada -SAM cells were untreated (UT) or treated with 1 μM Tg for 24 h, then immunoprecipitated with either an IgG control or a THADA antibody, followed by western blot analysis with a FADD antibody. ( i ) MIN6 beta-cell lines were treated with 1 μM Tg for 24 h, then immunoprecipitated with either an IgG control or a caspase-8 antibody, followed by western blot analysis with THADA and FADD antibodies. j Flow cytometry analysis of control and Thada -SAM β-cells treated with 1 μM Tg in the presence or absence of 20 μM Z-VAD or 20 μM Z-IETD for 24 h. Representative dot plots of cell apoptosis were shown after dual staining with Annexin V and 7-AAD. k The apoptosis of control and Thada -SAM β-cells were quantified by the percentage of Annexin V-positive cells ( n = 3). l The caspase-3/7 activities of control and Thada -SAM β-cells were assayed after treatment with or without 1 μM Tg in the presence or absence of 20 μM Z-VAD or 20 μM Z-IETD for 24 h ( n = 8 biologically independent samples). All western blots show representative results from at least three independent experiments. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; significance is assessed by two-way ANOVA followed by Tukey’s multiple comparison test ( a , l ), two-tailed unpaired Student’s t test ( c , d ), or one-way ANOVA followed by Dunnett’s multiple test ( k ). Source data are provided as a Source Data file.

Article Snippet: Stable MIN6 beta-cell line with endogenous Thada -activation was generated by CRISPR/dCas9-SAM system from Genechem (Shanghai, China).

Techniques: Control, Immunoprecipitation, Western Blot, Flow Cytometry, Staining, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: THADA inhibition in mice protects against type 2 diabetes mellitus by improving pancreatic β-cell function and preserving β-cell mass

doi: 10.1038/s41467-023-36680-0

Figure Lengend Snippet: a Body weights of WT ( n = 12) and Thada -KO mice ( n = 8) after HFHS diet for the indicated weeks. b Blood glucose levels of HFHS-fed WT ( n = 12) and Thada -KO mice that were fasted overnight ( n = 8). c Fed blood glucose levels of WT ( n = 12) and Thada -KO mice ( n = 8) after HFHS diet for 8 and 12 weeks. d Fed serum insulin levels of HFHS-fed WT and Thada -KO mice ( n = 7). e , f IPGTT and area under the curve of HFHS-fed WT ( n = 12) and Thada -KO mice ( n = 8). g , h ITT and area under baseline of HFHS-fed WT ( n = 7) and Thada -KO mice ( n = 8). i Serum insulin levels at 0, 15, and 30 min after intraperitoneal glucose injection in HFHS-fed WT and Thada -KO mice ( n = 8 for 0 min, n = 12 for 15 and 30 min). j Islets isolated from HFHS-fed WT ( n = 3) and Thada -KO mice ( n = 4) were stimulated with 3.3 and 16.7 mM glucose for 1 h, and insulin secretion was assayed. k Quantification of [Ca 2+ ] i in primary islet cells from NCD-fed WT, HFHS-fed WT and HFHS-fed Thada -KO mice at basal 2.8 mM glucose and after stimulation with 16.7 mM glucose ( n = 16 from five mice each group). l Quantification of [Ca 2+ ] i in primary islet cells from NCD-fed WT, HFHS-fed WT and HFHS-fed Thada -KO mice at basal 2.8 mM glucose and after stimulation with 35 mM KCl ( n = 16 from five mice each group). m Quantification of [Ca 2+ ] i in primary islet cells from NCD-fed WT, HFHS-fed WT and HFHS-fed Thada -KO at basal 5.6 mM glucose and after 10 μM ionomycin stimulation ( n = 14 for NCD-WT, n = 15 for HFHS-WT, n = 16 for HFHS-KO from five mice each group). n Measurements of β-cell area/pancreatic area ratio in HFHS-fed WT and Thada -KO mice ( n = 3). o Measurements of β-cell mass in HFHS-fed WT and Thada -KO mice ( n = 3). p Representative images of islets from HFHS-fed WT and Thada -KO mice stained for insulin (green), Tunel (red), and DAPI (blue). Scale bars, 50 μm. Arrowhead points to Tunel + Insulin + cells. The percentage of Tunel + in Insulin + cells was quantified ( n = 4 mice). At least 3000 Insulin + cells in each mouse were counted for quantification. q Representative images of islets from NCD-fed WT, HFHS-fed WT, and HFHS-fed Thada -KO mice stained for insulin (green), cleaved caspase-3 (red), and DAPI (blue). Scale bar was 20 or 50 μm as indicated. Arrowhead points to cleaved caspase-3 + Insulin + cells. The percentage of cleaved caspase-3 + in Insulin + cells was quantified ( n = 4 mice for NCD-WT and HFHS-WT, n = 3 mice for HFHS-KO). At least 2500 Insulin + cells in each mouse were counted for quantification. The experiments were performed on male mice after HFHS diet for 12 weeks unless otherwise indicated and female mice data were provided in Fig. . Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; significance is assessed by two-tailed unpaired Student’s t test (a– j , n – p ) or one-way ANOVA followed by Dunnett’s multiple comparison test ( k , l , m , q ). Source data are provided as a Source Data file.

Article Snippet: Stable MIN6 beta-cell line with endogenous Thada -activation was generated by CRISPR/dCas9-SAM system from Genechem (Shanghai, China).

Techniques: Injection, Isolation, Staining, TUNEL Assay, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: THADA inhibition in mice protects against type 2 diabetes mellitus by improving pancreatic β-cell function and preserving β-cell mass

doi: 10.1038/s41467-023-36680-0

Figure Lengend Snippet: a A high-content screening workflow to identify compounds that can reverse THADA-reduced [Ca 2+ ] i . b Primary screen data. Glucose-stimulated [Ca 2+ ] I was normalized to Z score. c Representative images of glucose-stimulated fluo-4 Ca 2+ fluorescence after treatment with vehicle or alnustone for 24 h. Scale bar, 100 μm. d Dose curve of alnustone on the glucose-stimulated [Ca 2+ ] i ( n = 3 biologically independent experiments). e GSIS of Thada -SAM β-cells treated with or without 10 μM alnustone for 24 h ( n = 4 biologically independent experiments). f MIN6 beta-cell lines were treated with or without 33 mM glucose and 0.5 mM palmitate (HGPA) in the presence or absence of 10 μM alnustone for 24 h. Then GSIS was assayed ( n = 4 biologically independent experiments). g – k DIO male mice were injected with vehicle or 10 mg/kg alnustone for 5–7 days, then body weights ( g ), fed blood glucose levels ( h ), IPGTT and the related AUC ( i , j ) as well as serum insulin levels at 0 and 15 min after intraperitoneal glucose injection ( k ) were measured ( n = 5 for vehicle and n = 7 for alnustone). l Model depicting the role of THADA in the pathogenesis of T2DM: THADA expression is induced by glucolipotoxicity in β-cells during T2DM progression, which leads to ER Ca 2+ depletion due to inhibited SERCA2 activity and leaky RyR2 Ca 2+ channel, and activation of the DR5/FADD/caspase-8 pro-apoptotic complex, consequently resulting in impaired insulin secretion and aggravated ER stress-induced β-cell apoptosis. Genetic ablation or inhibition of THADA by alnustone improves glucose tolerance in mice by promoting β-cell function and survival. Image was created with BioRender.com. K ATP : ATP-sensitive K + channel; L-VGCC: L-type voltage-gated Ca 2+ channel; ER: endoplasmic reticulum; SAM: synergistic activation mediator. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; significance is assessed by two-way ANOVA followed by Tukey’s multiple comparison test ( e ) or two-tailed unpaired Student’s t test ( f – k ). Source data are provided as a Source Data file.

Article Snippet: Stable MIN6 beta-cell line with endogenous Thada -activation was generated by CRISPR/dCas9-SAM system from Genechem (Shanghai, China).

Techniques: High Content Screening, Fluorescence, Injection, Expressing, Activity Assay, Activation Assay, Inhibition, Cell Function Assay, Comparison, Two Tailed Test

Journal: Oxidative Medicine and Cellular Longevity

Article Title: ∆nFGF1 Protects β -Cells against High Glucose-Induced Apoptosis via the AMPK/SIRT1/PGC-1 α Axis

doi: 10.1155/2022/1231970

Figure Lengend Snippet: ∆nFGF1 relieves β -cell apoptosis in db / db mice. (a) H&E staining of pancreas tissue from mice sacrificed after intraperitoneal (i.p.) injection of ∆nFGF1 or saline for 8 weeks (left panel). The black dotted lines indicate the borders of pancreatic islets. Pancreatic islet areas were quantified using ImageJ (right panel). (b) Coimmunofluorescence staining of insulin (green) and C-caspase 3 (red) in pancreatic islets. DAPI was used to stain nuclei. The white arrows indicate insulin/C-caspase 3 double-positive β -cells. (c) Representative PCNA-positive cells in pancreas sections (left panel). The black dotted lines indicate the borders of pancreatic islets. PCNA-positive cells were quantified using ImageJ (right panel). All data are presented as mean values ± SEM. n = 5 mice per group. ∗∗∗ p < 0.001; n.s.: no significance.

Article Snippet: Mouse pancreatic β -cell line MIN6 (Keygen Biotech, Nanjing, China) cells were cultured in RPMI1640 medium (Gibco) accompanied with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin.

Techniques: Staining, Injection, Saline

Journal: Oxidative Medicine and Cellular Longevity

Article Title: ∆nFGF1 Protects β -Cells against High Glucose-Induced Apoptosis via the AMPK/SIRT1/PGC-1 α Axis

doi: 10.1155/2022/1231970

Figure Lengend Snippet: ∆nFGF1 inhibits apoptosis of MIN6 cells. MIN6 cells were exposed to NG (11.1 mM) and HG+PA (33 mM HG+0.5 mM PA) in the presence or absence of ∆nFGF1 for 24 h. MIN6 cells were exposed for 24 h to HG or PA. (a) The expression of Bcl-2 was analyzed by Western blot (left panel) and quantified using ImageJ (right panel). (b) The expression of Bax and cleaved- (C-) caspase 3 in MIN6 cells under different concentrations of ∆nFGF1 was analyzed by Western blot (upper panel) and quantitated using ImageJ (lower panels). (c) The survival rate of MIN6 cells for above treatments. (d) The proliferation of MIN6 cells after treatment with FGF1 WT and ∆nFGF1. Representative immunofluorescence images of MIN6 cells stained with (e) TUNEL (green) and (f) C-caspase 3 (red). DAPI was used to stain nuclei. (g) The numbers of TUNEL-positive cells (upper panel) and the fluorescence intensity of C-caspase 3 were quantified using ImageJ (lower panel). (h) Bax, Bcl-2, and C-caspase 3 expression was analyzed by Western blot (left panel) and quantitated by ImageJ (right panels). β -Actin was used as a control. All data are presented as mean values ± SEM. NG: normal glucose; HG: high glucose; PA: palmitic acid. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. n.s.: no significance.

Article Snippet: Mouse pancreatic β -cell line MIN6 (Keygen Biotech, Nanjing, China) cells were cultured in RPMI1640 medium (Gibco) accompanied with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, TUNEL Assay, Fluorescence, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: ∆nFGF1 Protects β -Cells against High Glucose-Induced Apoptosis via the AMPK/SIRT1/PGC-1 α Axis

doi: 10.1155/2022/1231970

Figure Lengend Snippet: ∆nFGF1 induces activation of AMPK/Sitr1/PGC-1 α signaling. MIN6 cells were exposed to NG (11.1 mM) and HG+PA (33 mM HG+0.5 mM PA) in the absence or presence of ∆nFGF1 for 24 h. (a) The expression of phosphorylated- (p-) AMPK and AMPK analyzed by Western blot (left panel) and quantified by ImageJ (right panel) ( n = 3). (b) The expression levels of SIRT1 and PGC-1 α analyzed by Western blot (left panel) and quantified by ImageJ (right panel) ( n = 3). β -Actin was used as a loading control. (c, d) Immunostaining for SIRT1 (red) and PGC-1 α (green) in paraffin-embedded islets. DAPI was used to stain cell nuclei. n = 5 mice per group. (e) Quantitative analysis of fluorescence intensity of SIRT1 (upper panel) and PGC-1 α (lower panel). All data are presented as mean values ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Mouse pancreatic β -cell line MIN6 (Keygen Biotech, Nanjing, China) cells were cultured in RPMI1640 medium (Gibco) accompanied with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin.

Techniques: Activation Assay, Expressing, Western Blot, Control, Immunostaining, Staining, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: ∆nFGF1 Protects β -Cells against High Glucose-Induced Apoptosis via the AMPK/SIRT1/PGC-1 α Axis

doi: 10.1155/2022/1231970

Figure Lengend Snippet: ∆nFGF1 inhibits glucolipotoxicity-induced apoptosis in MIN6 cells via AMPK/SIRT1/PGC-1 α signaling. MIN6 cells were exposed to NG (11.1 mM) and HG+PA (33 mM HG+0.5 mM PA) with or without the AMPK inhibitor Compound C (CC) in the presence or absence of ∆nFGF1 for 24 h. (a) The protein levels of p-AMPK, SIRT1, and PGC-1 α analyzed by Western blot (left panel) and quantified by ImageJ (right panels) ( n = 3). (b) The protein expression levels of Bcl-2, Bax, and C-caspase 3 analyzed by Western blot (left panel) and quantified by ImageJ (right panels) ( n = 3). (c) MIN6 cells were transfected with AMPK siRNA for 48 h before exposure to NG (11.1 mM) and HG+PA (33 mM HG+0.5 mM PA) in the presence or absence of ∆nFGF1 for 24 h. The protein expression levels of p-AMPK, SIRT1, and PGC-1 α analyzed by Western blot (left panel) and quantified by ImageJ (right panels) ( n = 3). (d) The protein expression levels of Bcl-2, Bax, and C-caspase 3 analyzed by Western blot (left panel) and quantified by ImageJ (right panels) ( n = 3). β -Actin was used as a loading control. (e) Apoptosis of β -cells in each group was evaluated by flow cytometry analysis. The apoptotic rate was calculated as the percentage of Annexin V-positive cells divided by the total number of cells. (f) Schematic diagram illustrating the model of ∆nFGF1-mediated inhibition of pancreatic β -cell apoptosis in T2DM. All data are presented as mean values ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. n.s.: no significance.

Article Snippet: Mouse pancreatic β -cell line MIN6 (Keygen Biotech, Nanjing, China) cells were cultured in RPMI1640 medium (Gibco) accompanied with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin.

Techniques: Western Blot, Expressing, Transfection, Control, Flow Cytometry, Inhibition