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

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Effect of PRX-1 Downregulation in the Type 1 Diabetes Microenvironment

doi: 10.4196/kjpp.2012.16.6.463

Figure Lengend Snippet: Viral vector used in knockdown of PRX-1 in RAW264.7 and MIN6 cells.

Article Snippet: MIN6 cells were obtained from the Korean Cell Line Bank, RAW264.7 macrophage cell lines were obtained from the American Type Culture Collection (Manassas, USA) and both were maintained in DMEM medium (GIBCO) supplemented with 10% heat inactivated FBS (fetal bovine serum, CELLGRO), 2 mM L-glutamine, 100 U/ml penicillin and streptomycin (CELLGRO) at 37°C in a 5% CO 2 humidified incubator.

Techniques: Plasmid Preparation, Knockdown

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Effect of PRX-1 Downregulation in the Type 1 Diabetes Microenvironment

doi: 10.4196/kjpp.2012.16.6.463

Figure Lengend Snippet: (A) shPRX-1 construct and empty vector transduction efficiency. WT, scramble, and shPRX-1 1×10 6 MIN6 or RAW264.7 cells were harvested in PBS 1 ml. Then, the transduction efficiency was detected by phase fluorescence microscopy, representative imagery are shown. WT: wild type; Scramble: empty vector; shPRX-1: PRX-1-knockdown. (B) shPRX-1 silenced PRX-1 at gene level. Scramble, and shPRX-1 2×10 5 MIN6 or RAW264.7 cells were incubated in 24 well plate overnight. Total mRNA were extracted by Trizol and verified by RT-PCR, representative bands are shown. (C) shPRX-1 silenced PRX-1 at protein levels. Scramble, and shPRX-1 2×10 6 MIN6 or RAW264.7 cells were incubated in 6 well plate overnight. Whole protein was extracted by RIPA buffer and verified by western blot analysis, representative blots are shown.

Article Snippet: MIN6 cells were obtained from the Korean Cell Line Bank, RAW264.7 macrophage cell lines were obtained from the American Type Culture Collection (Manassas, USA) and both were maintained in DMEM medium (GIBCO) supplemented with 10% heat inactivated FBS (fetal bovine serum, CELLGRO), 2 mM L-glutamine, 100 U/ml penicillin and streptomycin (CELLGRO) at 37°C in a 5% CO 2 humidified incubator.

Techniques: Construct, Plasmid Preparation, Transduction, Fluorescence, Microscopy, Knockdown, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Effect of PRX-1 Downregulation in the Type 1 Diabetes Microenvironment

doi: 10.4196/kjpp.2012.16.6.463

Figure Lengend Snippet: Apoptotic gene expression in RAW264.7 and MIN6 co-cultures visualized through RT-PCR. (A) RAW264.7 cells were pre-stimulated with LPS for 4 hr before 24 hr of co-culture. Total RNA from each group was extracted and reverse transcribed for RT-PCR analysis. (B) Under the same conditions, L-NMMA was added to exclude the effect of NO and gene expression was assessed by RT-PCR.

Article Snippet: MIN6 cells were obtained from the Korean Cell Line Bank, RAW264.7 macrophage cell lines were obtained from the American Type Culture Collection (Manassas, USA) and both were maintained in DMEM medium (GIBCO) supplemented with 10% heat inactivated FBS (fetal bovine serum, CELLGRO), 2 mM L-glutamine, 100 U/ml penicillin and streptomycin (CELLGRO) at 37°C in a 5% CO 2 humidified incubator.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Co-Culture Assay, Reverse Transcription

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Effect of PRX-1 Downregulation in the Type 1 Diabetes Microenvironment

doi: 10.4196/kjpp.2012.16.6.463

Figure Lengend Snippet: NO production and cell viability of scramble and shPRX-1 MIN6 cells in the presence or absence of IL-6 and IL-10. (A) NO production of MIN6 cells were observed after treatment with an inflammatory cytokine mixture for 24 hr and the addition of IL-6, IL-10 or a combination of both. Values represent the mean±SD ( * , p<0.05 vs. scramble, ** , p<0.01 vs. scramble). (B) Cell viability of MIN6 cells were analyzed after treatment with an inflammatory cytokine mixture for 24 hr and in the presence or absence of IL-6. Values represent the mean±SD ( * , p<0.05 vs. scramble, ** , p<0.01 vs. scramble).

Article Snippet: MIN6 cells were obtained from the Korean Cell Line Bank, RAW264.7 macrophage cell lines were obtained from the American Type Culture Collection (Manassas, USA) and both were maintained in DMEM medium (GIBCO) supplemented with 10% heat inactivated FBS (fetal bovine serum, CELLGRO), 2 mM L-glutamine, 100 U/ml penicillin and streptomycin (CELLGRO) at 37°C in a 5% CO 2 humidified incubator.

Techniques:

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Effect of PRX-1 Downregulation in the Type 1 Diabetes Microenvironment

doi: 10.4196/kjpp.2012.16.6.463

Figure Lengend Snippet: The expression of iNOS, Bcl-xL and Bim was assessed by RT-PCR. Under cytokine mix treatment, IL-6 down-regulates iNOS expression in a significant manner in scramble MIN6 cells compared to shPRX-1 cells. shPRX-1 MIN6 cells treated with IL-6 showed increased expression of the anti-apoptotic gene, Bcl-xL and decreased expression of the pro-apoptotic gene, Bax.

Article Snippet: MIN6 cells were obtained from the Korean Cell Line Bank, RAW264.7 macrophage cell lines were obtained from the American Type Culture Collection (Manassas, USA) and both were maintained in DMEM medium (GIBCO) supplemented with 10% heat inactivated FBS (fetal bovine serum, CELLGRO), 2 mM L-glutamine, 100 U/ml penicillin and streptomycin (CELLGRO) at 37°C in a 5% CO 2 humidified incubator.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction