Journal: Frontiers in Immunology

Article Title: Oxidative Stress Leads to β-Cell Dysfunction Through Loss of β-Cell Identity

doi: 10.3389/fimmu.2021.690379

Figure Lengend Snippet: Oxidative stress leads to loss of β-cell maturity markers and a concomitant increase in progenitor marker expression. To evaluate the effect of oxidative stress on β-cell maturity markers and progenitor markers, primary human islets (blue graphs) or EndoC-βH1 cells (red graphs) were treated with hydrogen peroxide (H 2 O 2 ). (A) Oxidative stress induction in human islets by treatment with 200 μM H 2 O 2 for 90 minutes leads to decreased mRNA expression levels of the β-cell-specific genes MAFA, PDX1, KIR6.2, MAFB, FOXA2, PAX6, NKX2.2, NEUROD1 and GLUT1 as measured by qPCR. (B) The level of β-cell-specific protein MAFA is decreased in human islets upon treatment with 200 μM H 2 O 2 for 90 minutes as measured by Western blot. (C) Oxidative stress induction in EndoC-βH1 cells by treatment with 50 μM H 2 O 2 for 24 hours and 200 μM H 2 O 2 for 90 minutes leads to decreased mRNA expression levels of the β-cell-specific genes MAFA, PDX1, PAX4, NKX6.1, insulin, KIR6.2, MAFB, FOXA2, PAX6, NKX2.2, NEUROD1 and GLUT1 as measured by qPCR. (D) The level of β-cell-specific proteins MAFA and PDX1 is decreased in EndoC-βH1 cells upon treatment with 200 μM H 2 O 2 for 90 minutes as measured by Western blot. (E) Oxidative stress induction in EndoC-βH1 cells by treatment with 50 μM H 2 O 2 for 24 hours and 200 μM H 2 O 2 for 90 minutes leads to increased mRNA expression levels of the progenitor cell-specific genes SOX9 and HES1 as measured by qPCR. (F) The level of progenitor cell-specific protein SOX9 is increased in human islets upon treatment with 200 μM H 2 O 2 for 90 minutes as measured by Western blot. Data are presented as means ± SEM of fold change over untreated control islets (blue graphs) or EndoC-βH1 cells (red graphs). n=1-10 donors/batches; each data point represents one donor/batch. *p < 0.05, **p < 0.01, ***p < 0.0005, ****p < 0.0001 vs. untreated control islets/EndoC-βH1 cells as determined by a paired Student’s t test on the dCT values. Black circles = untreated control islets/EndoC-βH1 cells, upward-pointing triangles = H 2 O 2 -treated islets/EndoC-βH1 cells (50 μM 24h), downward-pointing triangles = H 2 O 2 -treated islets/EndoC-βH1 cells (200 μM 90min). h = hours, min = minutes, H 2 O 2 = hydrogen peroxide.

Article Snippet: Protein content in the supernatant was quantified using a BCA assay and immunoblotted with antibodies MAFA (Bethyl, A700-067), PDX1 (Abcam, AB47267) and SOX9 (Cell Signaling Technology, 82630S).

Techniques: Marker, Expressing, Western Blot, Control

Journal: Advanced Science

Article Title: Beta‐Cell Tipe1 Orchestrates Insulin Secretion and Cell Proliferation by Promoting Gαs/cAMP Signaling via USP5

doi: 10.1002/advs.202304940

Figure Lengend Snippet: Sequences of real‐time PCR primers.

Article Snippet: Slides were incubated with the appropriate antibodies against TIPE1 (#BS60529, Bioworld technology), PCNA (#ab92552, Abcam), MAFA (#sc‐390491, Santa Cruz Biotechnology), PDX1 (#sc‐390792, Santa Cruz Biotechnology), GLUT2 (#sc‐518022, Santa Cruz Biotechnology), Gαs (#sc‐55545, Santa Cruz Biotechnology), USP5 (#sc‐390943, Santa Cruz Biotechnology), HA (#M180‐3, MBL), DDDK (#M185‐3, MBL), Myc (#18 583, CST), GFP (#66002‐1‐Ig, Proteintech), GASP1 (#A16090, ABclonal), PKA (#AF7746, Affinity), p‐PKA (Thr198) (#AF7246, Affinity), CREB (#9197S, CST), p‐CREB (Ser133) (#9198, CST), AMPK (#ab80039, Abcam), P‐AMPK (Thr172) (#2535S, CST), AKT (#ab18785, abcam), p‐AKT (S473) (#4058s, CST), p‐AKT (T308) (#13038S, CST), IR (#sc57342, Santa Cruz Biotechnology), p‐IR (Tyr1150/1151) (#sc‐81500, Santa Cruz Biotechnology), Caspase 3 (#14 220, CST), Caspase 9 (#9508S, CST), Bcl‐2 (#ab182858, Abcam), Ub (#3936, CST), Ub‐K48 (#4289S, CST), Ub‐K63 (#5621S, CST), MAEA (#abs133634, Absin Biotechnology), WWP2 (#sc‐398090, Santa Cruz Biotechnology), USP9X (#sc‐365353, Santa Cruz Biotechnology), β‐actin (#66009‐1‐Ig, Proteintech), and GAPDH (#60004‐1‐Ig, Proteintech).

Techniques: Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: T cell receptor signaling strength establishes the chemotactic properties of effector CD8 + T cells that control tissue-residency

doi: 10.1038/s41467-023-39592-1

Figure Lengend Snippet: a Naïve IFNγ-YFP P14 CD8 + T cells were transferred into B6 mice and infected on the ear skin with VacV-GP33. YFP expression in the indicated organs was quantified by flow cytometry on day 7 post infection. b Quantification of ( a ) at the indicated timepoints after infection; n = 3 per tissue per time point. c Quantification of the total number and YFP + P14 CD8 + T cells over time; n = 3. d Same as ( a ) except CD8β antibody was injected intravenously before sacrifice to distinguish YFP-P14 CD8 + T cells within the skin from those in the vasculature. e Quantification of ( d ); n = 3. f Same as ( a ) except YFP expression was quantified by P14 CD8 + T cells in the dermis and epidermis on day 7 post infection. g Quantification of ( f ); n = 4. h Experiment design for ( i – r ). i Representative flow plots depicting the total number of P14 CD8 + T cells and YFP expression by IFNγ-YFP P14 CD8 + T in the skin on day 7 post infection. j Quantification of the total number of P14 CD8 + T cells in ( i ); n = 12. k Quantification of YFP expression in ( i ); n = 12. l Representative histograms depicting the expression of KLRG1. m Quantification of ( l ); n = 3. n Representative flow plots depicting the expression of YFP and KLRG1. o Quantification of ( n ); n = 3. p Quantification of the total number of IFNγ-YFP P14 CD8 + T cells in the skin on day 40 post infection; n = 5. q Representative flow plots depicting the expression of CD69 and CD103 on day 40 post infection. r Quantification of ( q ); n = 5. Data shown are mean ± SD and representative of 2 or more independent experiments except (j,k) which are cumulative data from 3 independent experiments. Statistical significance was calculated using a paired two-sided t-test ( e , g , k , o , p , r ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( m ). Source data are provided as a Source Data file.

Article Snippet: The following antibodies along with appropriate isotype controls were used in this study: CD45.2 PE/Cyanin-7, Pacific Blue or APC (1:400; Clone 104; BioLegend; Cat# 109830, 109820 or 109814), CD8α Brilliant Violet 711, Pacific Blue, APC, or BUV395 (1:400; Clone 53-6.7; BioLegend; Cat# 100759, 100725, 100711, or BD Biosciences Cat# 563786), CD44 Pacific Blue (1:400; Clone IM7; BioLegend; Cat# 103020), Thy1.1 PerCP/Cy5.5, Brilliant Violet 711, or Pacific Blue (1:1000; Clone OX7; BioLegend; Cat# 109004, 202539 or 202522), Thy1.2 Brilliant Violet 605 or Pacific Blue (1:1000; clone 53-2.1; BioLegend; Cat# 140318 or 140306), KLRG1 Violet Fluor 450 (1:200; Clone 2F1; Tonbo; Cat# 75-5893-U100), CD8β PerCP/Cy5.5 (Clone YST156.7.7; BioLegend; Cat# 126609), CD69 FITC or Pacific Blue (1:100; Clone H1.2FE, BioLegend; Cat# 104506 or 104523), CD103 PE (1:200; Clone 2-E7; BioLegend; Cat# 121406), IFNγ APC (1:200; Clone XMG1.2; BioLegend; Cat# 505810), PD-1 PE (1:100; Clone 29F1.1a12; BioLegend; Cat# 135206), ICOS FITC or PE (1:200; Clone 7E.17G9, eBioscience; Cat# 11-9942-82 or 12-9942-82), CXCR6 APC (1:300; Clone SA051D1, BioLegend; Cat# 151106), Blimp1 PE (1:200; Clone 5-E7, BioLegend; Cat# 150006), TCF-1 PE (1:200; Clone S33-966; BD Bioscience; Cat# 564217), Ki-67 PE/Cyanin7 (1:200; Clone 16A8, BioLegend; Cat# 652425), CD62L PE or APC (1:400; Clone MEL-14; BioLegend; Cat# 104408 or 104412), CD25 PE (1:200; Clone PC61; BioLegend; Cat# 102007) and Viability-Ghost Dye Red 780 (1:1000; Tonbo; Cat #13-0865-T100).

Techniques: Infection, Expressing, Flow Cytometry, Injection

Journal: Bioresources and bioprocessing

Article Title: Effect of Akkermansia muciniphila on pancreatic islet β-cell function in rats with prediabetes mellitus induced by a high-fat diet.

doi: 10.1186/s40643-024-00766-4

Figure Lengend Snippet: Fig. 2 Effects of A. muciniphila treatment and dietary intervention on GSIS, apoptosis and differentiation in the β cells of rats with pre-DM: A GSIS index in the β cells of rats in different groups; B TUNEL and insulin immunofluorescence staining (×400) of the pancreatic tissues of rats in different groups (blue fluorescence is nuclear dye [DAPI] staining, red fluorescence represents insulin-positive β cells, green fluorescence represents TUNEL-positive apoptotic cells, and Merge is TUNEL combined with insulin immunofluorescence staining); C Comparison of the percentage of TUNEL+ β cells of rats in different groups; D-I Comparison of the expression of Bcl-2, Bax, Bcl-2/Bax, Caspase3, MAFA and MAFB at the mRNA level in the islets of rats in different groups; J Comparison of the expression of MAFA and MAFB at the protein level in the islets of rats in different groups. Different letters in the same bar represent significant differences between the treatments when P < 0.05

Article Snippet: with protein-free rapid blocking buffer (PS108P, Epizyme Biotech, Shanghai, China) at room temperature for 1 h, the membranes were incubated with different primary antibodies at 4 °C overnight, such as a MAFA rabbit monoclonal antibody (mAb) (1:1000, 79,737, Cell Signaling Technology, Danvers, MA, USA), a MAFB rabbit mAb (1:1000, 30,919, Cell Signaling Technology, Danvers, MA, USA), an Occludin mAb (1:1000, 33-1500, Invitrogen, Carlsbad, CA, USA), an anti-TLR2 antibody (1:500, ab209217, Abcam, Cambridge, UK), an anti-TLR4 antibody (1:500, ab13867, Abcam, Cambridge, UK), a GAPDH rabbit mAb (1:5000, 5174, Cell Signaling Technology, Danvers, MA, USA), and a beta actin polyclonal antibody (1:5000, 20536-1-AP, Proteintech, Chicago, IL, USA).

Techniques: TUNEL Assay, Immunofluorescence, Staining, Fluorescence, Comparison, Expressing