Journal: Cell reports

Article Title: Pancreatic islet protection at the expense of secretory function involves serine-linked mitochondrial one-carbon metabolism

doi: 10.1016/j.celrep.2023.112615

Figure Lengend Snippet: (A) Schematic of RNA-seq setup. Genes, differentially expressed after treatment of islets with 10 μM DXO for 24 h (to observe the initial alterations in gene expression, followed by protein-based and functional analyses at 48 h), are shown on the pie chart. Change of expression was calculated as log2 fold change (log2 FC) with cutoff ≥ 1 and ≤ −1, p value <0.05, fragments per kilobase million (FPKM) >1. (B) Expression analysis of ER stress marker genes. (C and D) (C) Atf4 immunoblot of mouse pancreatic islets treated with 10 μM DXO for 48 h, and (D) quantification; n = 7 independent experiments. (E) Expression analysis of genes encoding for enzymes of de novo serine synthesis. (F and G) (F) Phgdh immunoblot of mouse islets treated with 10 μmM DXO for 48 h, and (G) quantification; n = 5 independent experiments. (H) Expression analysis of genes encoding for enzymes involved in the mitochondrial and cytosolic OCM. (I) Aldh1l2 immunoblot with fractionated (upper blot) and whole (lower blot) lysates of mouse islets treated with 10 μM DXO for 48 h. (J) Quantification of whole-cell lysates; n = 12 independent experiments. Beta-actin was used for normalization. Graphs show genes and their log2 FC, FPKM, and p values based on RNA-seq data. The size of the circles is inversely proportional to the p value. Horizontal axes show FPKM values, vertical axes show log2 FC. Dashed lines indicate the restriction criteria of the FPKM and the log2 FC. Gradients on the left indicate the fold change. Genes of the Atf4 activated branch and cytosolic/mitochondrial serine-linked OCM are highlighted by larger font size. All quantifications of blots are shown as percentage of control, and significance was determined by two-tailed paired Student’s t test (D, G, J). See also – for islet cell and proliferation marker gene expression levels.

Article Snippet: For efficient overexpression of ATF4 in pancreatic islets, dispersed islet cells were infected for 6 h in a humidified incubator at 37°C and 5% CO 2 with a multiplicity of infection (MOI) of 200 with adenoviruses containing either a human ATF4 overexpression plasmid (Vector Biolabs, # ADV-201618, RefSeq: BC011994, further designated as Ad- ATF4 ) or a GFP control plasmid (ViraQuest Inc., VQAd CMV eGFP, further designated as Ad- GFP ), followed by the formation of pseudo-islets by hanging drop technique.

Techniques: RNA Sequencing, Gene Expression, Functional Assay, Expressing, Marker, Western Blot, Control, Two Tailed Test

Journal: Cell reports

Article Title: Pancreatic islet protection at the expense of secretory function involves serine-linked mitochondrial one-carbon metabolism

doi: 10.1016/j.celrep.2023.112615

Figure Lengend Snippet: (A) Immunoblot for Xbp-1s (a marker of unfolded protein response, UPR in brief) in islets after treatment with 10 μM DXO for 24 h. (B) Quantification of Xbp-1s normalized to Beta-actin; n = 4 independent treatments. (C–J) Islets were left untreated (control), treated with 10 μM DXO for 72 h, or treated with 10 μM DXO for 48 h followed by 24 h washout. (C) Immunoblot for Aldh1l2, Atf4, and Beta-actin in lysates from pancreatic islets. Reproduced in five independent experiments. (D–J) Relative mRNA expression of (D) Atf4 , (E) Phgdh , (F) Psat1 , (G) Psph , (H) Shmt2 , (I) Mthfd2 , and (J) Aldh1l2 , in pancreatic islets. n = 4 independent experiments. Data are shown as mean ± SEM with corresponding p values and quantifications shown as percentage of control. Statistical significance was determined by two-tailed paired Student’s t test (B) and one-way ANOVA followed by Tukey’s multiple comparison test (D–J).

Article Snippet: For efficient overexpression of ATF4 in pancreatic islets, dispersed islet cells were infected for 6 h in a humidified incubator at 37°C and 5% CO 2 with a multiplicity of infection (MOI) of 200 with adenoviruses containing either a human ATF4 overexpression plasmid (Vector Biolabs, # ADV-201618, RefSeq: BC011994, further designated as Ad- ATF4 ) or a GFP control plasmid (ViraQuest Inc., VQAd CMV eGFP, further designated as Ad- GFP ), followed by the formation of pseudo-islets by hanging drop technique.

Techniques: Western Blot, Marker, Control, Expressing, Two Tailed Test, Comparison

Journal: Cell reports

Article Title: Pancreatic islet protection at the expense of secretory function involves serine-linked mitochondrial one-carbon metabolism

doi: 10.1016/j.celrep.2023.112615

Figure Lengend Snippet: (A) Scheme of adenoviral GFP (Ad- GFP ) and human ATF4 (Ad- ATF4 ) plasmid transduction into dispersed mouse islet cells and subsequent generation of pseudo-islets. Representative images of pseudo-islets infected with Ad- GFP . Scale bar, 100 μm. (B–H) Relative mRNA expression of (B) human ATF4 , (C) Phgdh , (D) Psat1 , (E) Psph , (F) Shmt2 , (G) Mthfd2 , and (H) Aldh1l2 in pseudo-islets infected with Ad- GFP or Ad- ATF4 . Reproduced in one (C and E) and three (D and F–H) independent experiments. (I) Immunoblot of Aldh1l2, Phgdh, ATF4, Beta-actin, and GFP in pseudo-islets infected with Ad- GFP as control or Ad- ATF4 . Relative protein expression was normalized to GFP expression levels. Reproduced in two independent experiments. (J) Insulin secretion of Ad- GFP and Ad- ATF4 infected pseudo-islets, after low (2 mM) and high (20 mM) glucose stimulation. Reproduced in two independent experiments. (K) Insulin content of infected pseudo-islets. Reproduced in two independent experiments. (L) Flow cytometry analysis of cell viability using FVS660. Histograms of Ad- GFP or Ad- ATF4 infected mouse islet cells, demonstrating live (FL4-H negative) and dead (FL4-H positive) cell populations after treatment with 2.5 mM STZ for 24 h. Reproduced in two independent experiments. (M) Bright-field images of infected pseudo-islets after 24 h of 2.5 mM STZ treatment. Scale bar, 100 μm. Please note that each dot in the insulin secretion and content panels represents an independent well containing seven or eight islets each, and data are presented as mean ± SEM with corresponding p values and quantifications shown as percentage of Ad- GFP control. Statistical significance was evaluated by two-tailed unpaired Student’s t test (B–H and K) or two-way ANOVA followed by Tukey’s multiple comparison test (J).

Article Snippet: For efficient overexpression of ATF4 in pancreatic islets, dispersed islet cells were infected for 6 h in a humidified incubator at 37°C and 5% CO 2 with a multiplicity of infection (MOI) of 200 with adenoviruses containing either a human ATF4 overexpression plasmid (Vector Biolabs, # ADV-201618, RefSeq: BC011994, further designated as Ad- ATF4 ) or a GFP control plasmid (ViraQuest Inc., VQAd CMV eGFP, further designated as Ad- GFP ), followed by the formation of pseudo-islets by hanging drop technique.

Techniques: Plasmid Preparation, Transduction, Infection, Expressing, Western Blot, Control, Flow Cytometry, Two Tailed Test, Comparison

Journal: Cell reports

Article Title: Pancreatic islet protection at the expense of secretory function involves serine-linked mitochondrial one-carbon metabolism

doi: 10.1016/j.celrep.2023.112615

Figure Lengend Snippet: (A–H) (A) Experimental setup for siRNA-mediated knockdown of Atf4 (si- Atf4 ) in dispersed mouse islet cells and generation of pseudo-islets. Scale bar, 100 μm. Relative mRNA expression of (B) Atf4 (n = 10) in control siRNA (si-Ctrl) and si- Atf4 transfected pseudo-islets, and relative mRNA levels of the enzymes (C) Phgdh , (D) Psat1 , (E) Psph , (F) Shmt2 , (G) Mthfd2 , and (H) Aldh1l2 in transfected pseudo-islets with or without 10 μM DXO for 48 h. Reproduced in three (G) and seven (C–F and H) independent experiments. (I) Immunoblot of Aldh1l2, Atf4, and Beta-actin in siRNA-transfected pseudo-islets that were either untreated or treated with 1 μM ER stressor thapsigargin (Thapsi) for 24 h. Beta-actin was used as a loading control. Reproduced in one independent experiment. (J) Insulin secretion of siRNA-transfected pseudo-islets treated with or without 10 μM DXO for 48 h, after low (2 mM) and high (20 mM) glucose stimulation. Reproduced in three independent experiments. (K) Insulin content in transfected pseudo-islets treated with or without 10 μM DXO for 48 h. Reproduced in three independent experiments. (L and M) (L) Quantification and (M) images of cell viability assays with siRNA-transfected pseudo-islets stained with EthD-1, calcein and Hoechst. Untreated, no DXO or STZ; 24 h 1.5 mM STZ, no DXO, followed by 24 h of 1.5 mM STZ; 10 μM DXO + 1.5 mM STZ, 24 h of 10 μM DXO, followed by 24 h of 1.5 mM STZ and 10 μM DXO. Scale bar, 50 μm. Please note that each dot in the insulin secretion and content panels represents an independent well containing seven or eight islets each; data are presented as mean ± SEM with corresponding p values, and quantifications are shown as percentage of untreated si- Ctrl . Statistical significance was determined by two-tailed paired Student’s t test (B) and two-way ANOVA, followed by Tukey’s multiple comparison test (C–H and J–L).

Article Snippet: For efficient overexpression of ATF4 in pancreatic islets, dispersed islet cells were infected for 6 h in a humidified incubator at 37°C and 5% CO 2 with a multiplicity of infection (MOI) of 200 with adenoviruses containing either a human ATF4 overexpression plasmid (Vector Biolabs, # ADV-201618, RefSeq: BC011994, further designated as Ad- ATF4 ) or a GFP control plasmid (ViraQuest Inc., VQAd CMV eGFP, further designated as Ad- GFP ), followed by the formation of pseudo-islets by hanging drop technique.

Techniques: Knockdown, Expressing, Control, Transfection, Western Blot, Staining, Two Tailed Test, Comparison

Journal: Cell reports

Article Title: Pancreatic islet protection at the expense of secretory function involves serine-linked mitochondrial one-carbon metabolism

doi: 10.1016/j.celrep.2023.112615

Figure Lengend Snippet:

Article Snippet: For efficient overexpression of ATF4 in pancreatic islets, dispersed islet cells were infected for 6 h in a humidified incubator at 37°C and 5% CO 2 with a multiplicity of infection (MOI) of 200 with adenoviruses containing either a human ATF4 overexpression plasmid (Vector Biolabs, # ADV-201618, RefSeq: BC011994, further designated as Ad- ATF4 ) or a GFP control plasmid (ViraQuest Inc., VQAd CMV eGFP, further designated as Ad- GFP ), followed by the formation of pseudo-islets by hanging drop technique.

Techniques: Virus, Control, Plasmid Preparation, Recombinant, Reverse Transcription, Fractionation, Enzyme-linked Immunosorbent Assay, Software, Staining