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: PLoS ONE

Article Title: Identification of Multiple Subsets of Ventral Interneurons and Differential Distribution along the Rostrocaudal Axis of the Developing Spinal Cord

doi: 10.1371/journal.pone.0070325

Figure Lengend Snippet: List of the primary antibodies used in this study.

Article Snippet: Rabbit anti-MafA , 1∶500 , Novus Biological , NB400-137.

Techniques: