Journal: Cell reports

Article Title: Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells

doi: 10.1016/j.celrep.2023.113615

Figure Lengend Snippet: (A) Ribosome (Ribo-seq) and RNA (RNA-seq) coverage plots for Atf4 mRNA in eIF2α +/+ and eIF2α A/A mESCs under LIF conditions. (B) The percentage of Atf4 mRNAs in each fraction of polysome profiles (left) and total input mRNA level (right) of eIF2α +/+ and eIF2α A/A mESCs cultured in LIF medium were quantified by RT-qPCR. *p < 0.05, (n = 3), two-way ANOVA test with correction for multiple comparisons using the Benjamini-Hochberg method. Data presented as mean ± SEM. (C) Ribo-seq and RNA-seq coverage plot of Epop in eIF2α +/+ and eIF2α A/A mESCs under LIF conditions. (D) The percentage of Epop mRNAs in each fraction of polysome profiles (left) and total input mRNA level (right) of eIF2α +/+ and eIF2α A/A mESCs cultured in LIF medium were quantified by RT-qPCR. *p < 0.05, **p < 0.01, (n = 3), two-way ANOVA test with correction for multiple comparisons using the Benjamini-Hochberg method. Data presented as mean ± SEM. (E) Position and amino acid conservation of Epop uORF-encoded protein in several mammals. The number of nucleotides (nt) between the cap and the start of uORF is indicated in blue for each species. The distance of the uORF start site to the CDS start site, and the length of the uORF-encoded protein are indicated in red and green, respectively, for each species. (F) Amino acid conservation of N-terminal region of EPOP uORF-encoded peptide in mammalian species. (G) Conservation of nucleotides flanking the start site of Epop uORF in mammalian species. (H) Left: UCSC Genome browser views for ATF4 binding events in GCN2 +/+ wild-type (WT) and GCN2 −/− knockout (KO) cells that were exposed to control (+Leu) or leucine-deficient (−Leu) medium. ATF4 binding profiles in ATF4 +/+ (WT) and ATF4 −/− (KO) cells treated with tunicamycin (Tm) (GSE35681) are shown below the panel. Right: the heatmap shows fold enrichment for ATF4 ChIP-seq target eIF1. (I and J) RT-qPCR analysis of N2A cells transduced with the indicated siRNA or shRNAs. Values were normalized to β-actin level. *p < 0.05, **p < 0.01. Data are presented as mean ± SEM (n = 3) t test. (K and L) Western blot analysis and quantification of ATF4 and eIF1 in N2A cells transduced with shCtrl or shRNA against ATF4. Values were normalized to β-actin level. Results are presented as mean ± SEM (n = 3). *p < 0.05, t test.

Article Snippet: Mouse monoclonal ATF4 (Clone B-3) , Santa Cruz Biotechnology , Cat#sc-390063; RRID:AB_2810998.

Techniques: RNA Sequencing, Cell Culture, Quantitative RT-PCR, Binding Assay, Knock-Out, Control, ChIP-sequencing, Transduction, Western Blot, shRNA

Journal: Cell reports

Article Title: Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells

doi: 10.1016/j.celrep.2023.113615

Figure Lengend Snippet: (A) Heatmap plot highlighting translationally downregulated mRNA involved in glutathione synthesis (eIF2α A/A compared with eIF2α +/+ mESCs under LIF conditions). Values are log-transformed z-scored TE. (B) Ribo-seq and RNA-seq coverage plot of Slc25a39 in eIF2α +/+ and eIF2α A/A mESCs under LIF conditions. (C) Position and amino acid conservation of Slc25a39 uORF-encoded protein in several mammals. The distance of the uORF stop codon to the CDS start codon (in red) and the length of the uORF-encoded peptide (in green) have been shown for each species. (D) Amino acid conservation of N-terminal region of Slc25a39 uORF-encoded peptides in different mammalian species (top), conservation of nucleotides around the start site of Slc25a39 uORF in different mammalian species (bottom). (E) Heatmap plot highlighting transcriptionally regulated genes involved in glutathione synthesis (eIF2α A/A compared with eIF2α +/+ mESCs in LIF). Values are log-transformed z-scored RNA-seq expression level. (F) UCSC Genome browser views for ATF4 binding events in GCN2 +/+ (WT) and GCN2 −/− (KO) cells that have been exposed to control (+Leu) or leucine-deficient (−Leu) medium. ATF4 binding profiles in ATF4 +/+ (WT) and ATF4 −/− (KO) cells treated with tunicamycin (Tm) (GSE35681) are shown below each panel. (G) The heatmap plot shows fold enrichment for selected ATF4 ChIP-Seq target genes associated with glutathione synthesis. (H) Metabolomic analysis of eIF2α +/+ and eIF2α A/A mESCs under LIF conditions. Significantly up- and downregulated metabolites (p < 0.05 and −0.5 > log 2 FC > 0.5) are highlighted in red and blue, respectively. (I) Schematic summary of genes that are translationally or transcriptionally regulated by ISR and are involved in glutathione metabolism. CTH, cystathionine gamma-lyase; CHAC1, ChaC glutathione-specific gamma-glutamylcyclotransferase 1; GPX, glutathione peroxidase; GSS, glutathione synthetase; GSSG, glutathione disulfide; GSH, glutathione; Glu, glutamate; Gln, glutamine.

Article Snippet: Mouse monoclonal ATF4 (Clone B-3) , Santa Cruz Biotechnology , Cat#sc-390063; RRID:AB_2810998.

Techniques: Transformation Assay, RNA Sequencing, Expressing, Binding Assay, Control, ChIP-sequencing

Journal: Cell reports

Article Title: Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells

doi: 10.1016/j.celrep.2023.113615

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal ATF4 (Clone B-3) , Santa Cruz Biotechnology , Cat#sc-390063; RRID:AB_2810998.

Techniques: Recombinant, Mass Spectrometry, SYBR Green Assay, Reverse Transcription, Phospho-proteomics, Derivative Assay, Plasmid Preparation, Control, Software

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: HFD feeding upregulates Adam10 and Adam17 expression in monocyte/macrophage populations of GWAT (A) Immunoblot analysis of ADAM10 and ADAM17 protein expression in gonadal white adipose tissue (GWAT) of mice after 8 weeks of NCD or HFD feeding ( n = 6 mice per group). (B) Umap plot of Adam10 , Adam17 , and total nuclei isolated from GWAT of NCD and HFD-fed mice (PRJNA942977). Clusters are colored by cell types: adipocyte, mesothelial cell, lymphatic endothelial cell, vascular endothelial cell, adipocyte progenitor cell, smooth muscle cell (SMC), monocyte/macrophage (mono/mac), dendritic cell (DC), B cell, and T cell. (C) Violin plot of Adam10 and Adam17 gene expression levels in total cell types from GWAT of NCD- and HFD-fed mice. (D) Umap plot of Adam10 , Adam17 from monocyte/macrophage population in GWAT of NCD- and HFD-fed mice (PRJNA942977). Clusters are colored by cell types: Lyve1 +macrophage (Mac. Lyve1 ), Trem2 +macrophage (Mac. Trem2 ), Prg4 +macrophage (Mac. Prg4 ), and monocyte. (E) Bubble plot and violin plot of Adam10 and Adam17 expression levels in distinct cell populations from mouse GWAT, determined by single-nucleus RNA sequencing (PRJNA942977). (F) qPCR analysis of Adam10 and Adam17 mRNA expression in isolated F4/80+ macrophages and adipocytes from GWAT after 8 weeks of NCD or HFD feeding ( n = 3 mice per group). Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: ADAM10 (B-3) Mouse mAb , Santa Cruz Biotechnology , Cat# sc-28358; RRID: AB_626636.

Techniques: Expressing, Western Blot, Isolation, Gene Expression, RNA Sequencing

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: Obesity increases ADAM10 and ADAM17 expression in macrophage populations of WAT (A) ADAM10 and ADAM17 expression levels in total cell types of human visceral adipose tissues ( GSE176171 ). Clusters are colored by cell types: Adipocyte, adipose stem and progenitor cells (ASPC), mesothelium, endothelial cell, lymphatic endothelial cell (LEC), pericyte, smooth muscle cell (SMC), macrophage, monocyte, dendritic cell (DC), mast cell, neutrophil, B cell, natural killer cell (NK cell), T cell, and endometrium cell. (B) Bubble plot and violin plot of ADAM10 and ADAM17 gene expressions and representative markers in adipocyte and macrophage populations with body mass index (BMI) in human subcutaneous adipose tissue ( GSE176171 ). (C) Violin plot of TREM2 , ADAM10 , and ADAM17 gene expression levels in human macrophage sub-populations (hMac1, hMac2, hMac3). (D) Correlation analysis of ADAM10 and ADAM17 gene expression levels with BMI in human subcutaneous adipose tissue ( n = 12 patients per group). Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: ADAM10 (B-3) Mouse mAb , Santa Cruz Biotechnology , Cat# sc-28358; RRID: AB_626636.

Techniques: Expressing, Gene Expression

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: Apoptotic adipocytes fail to induce TREM2 shedding in RAW264.7 cells and bone marrow-derived macrophages (BMDMs) (A) Schematic diagram illustrating the experimental method used for co-culturing dying/dead adipocytes with RAW264.7 cells or BMDMs. Apoptotic adipocytes (aAC) were generated by treating differentiated 3T3-L1 adipocytes with brefeldin A (BFA; 5 μg/mL for 24 h). Apoptotic adipocytes (1 × 10 5 cells/well) were directly co-cultured with RAW264.7 cells or BMDMs (5 × 10 5 cells/well) in growth medium. After 24 h of co-culture, non-engulfed or floating apoptotic adipocytes were removed by PBS washing, and RAW264.7 cells or BMDMs were subsequently harvested for immunoblot analysis. (B) Immunoblot analysis of caspase-3 expression levels in 3T3-L1 adipocytes after BFA for 24 h ( n = 3 cells per group). (C) and (D) Immunoblot analysis of TREM2, ADAM10, and ADAM17 protein expression levels in RAW264.7 cells (C) and BMDMs (D) co-cultured with apoptotic adipocytes ( n = 3 cells per group). Black arrow and red arrow indicate each precursor form of ADAM10/17 and active form of ADAM10/17. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: ADAM10 (B-3) Mouse mAb , Santa Cruz Biotechnology , Cat# sc-28358; RRID: AB_626636.

Techniques: Derivative Assay, Generated, Cell Culture, Co-Culture Assay, Western Blot, Expressing

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: GM6001 blocks TREM2 shedding in RAW264.7 cells induced by pyroptotic adipocytes (A) A schematic diagram illustrating the experimental method used for co-culturing pyroptotic adipocytes with macrophages. Pyroptotic adipocytes were generated by treating differentiated 3T3L1 adipocytes with lipopolysaccharide (LPS; 100 μg/μL, 48 h) followed by ATP (2 mM, 24 h). Pyroptotic adipocytes (1 × 10 5 cells/well) were directly co-cultured with RAW264.7 cells (5 × 10 5 cells/well) or BMDMs for 24 h in growth medium. After co-culture, non-engulfed or floating pyroptotic adipocytes were removed by PBS washing, and RAW264.7 cells and BMDMs were subsequently harvested for immunoblot analysis. (B) Immunoblot analysis of NLRP3, caspase-1, and GSDMD (gasdermin D) expression levels in 3T3-L1 adipocytes after LPS priming for 48 h and ATP treatment for 24 h ( n = 3 cells per group). Black arrow and red arrow indicate each total form of GSDMD and cleaved GSDMD. (C) Immunoblot analysis of TREM2, ADAM10, and ADAM17 expression levels in RAW264.7 cells co-cultured with pyroptotic adipocytes for 24 h in the presence or absence of GM6001 ( n = 4 cells per group). Black arrow and red arrow indicate each precursor form of ADAM10/17 and active form of ADAM10/17. (D) Immunoblot analysis of P-STING and STING expression levels in RAW264.7 cells co-cultured with pyroptotic adipocytes ( n = 3 cells per group). (E) Immunoblot analysis of P-syk, syk, P-PI3K, PI3K, P-PLCγ1, PLCγ1, P-AKT, and AKT protein levels in RAW264.7 cells co-cultured with pyroptotic adipocytes ( n = 3 cells per group). (F) Phagocytosis analysis of RAW264.7 cells co-cultured with pyroptotic adipocytes for 18 h in the presence of GM6001. Adipocytes were tagged with C12-BODIPY (red), and macrophages were stained with DiO (green). Representative images from three independent experiments are shown, with quantification provided in the right panel. Scale bars, 100 μm. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: ADAM10 (B-3) Mouse mAb , Santa Cruz Biotechnology , Cat# sc-28358; RRID: AB_626636.

Techniques: Generated, Cell Culture, Co-Culture Assay, Western Blot, Expressing, Staining

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: GM6001 prevents the cleavage of TREM2 on BMDMs induced by pyroptotic adipocytes (A) Immunoblot analysis of TREM2, ADAM10, and ADAM17 expression levels in BMDMs co-cultured with pyroptotic adipocytes for 24 h in the presence or absence of GM6001 ( n = 3 cells per group). Black arrow and red arrow indicate each precursor form of ADAM10/17 and active form of ADAM10/17. (B) and (C) Phagocytosis analysis of BMDMs, (B) TREM2 knockdown BMDMs, and negative control (NC). (C) co-cultured with pyroptotic adipocytes for 18 h in the presence of GM6001. Adipocytes were tagged with C12-BODIPY (red), and macrophages were stained with DiO (green). Representative images from three independent experiments are shown, with quantification provided in the right panel. The yellow boxes indicate the regions shown in the magnified images. Scale bars, 200 μm. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: ADAM10 (B-3) Mouse mAb , Santa Cruz Biotechnology , Cat# sc-28358; RRID: AB_626636.

Techniques: Western Blot, Expressing, Cell Culture, Knockdown, Negative Control, Staining

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: GM6001 treatment reduces TREM2 shedding and HFD-induced inflammation in GWAT (A) Schematic illustration of the experimental strategy for GM6001 administration (7.5 mg/kg/2 days) in mice fed a high-fat diet (HFD) for 10 weeks. (B) Immunoblot analysis of TREM2, ADAM10, and ADAM17 protein levels in gonadal white adipose tissue (GWAT) of HFD-fed mice treated with or without GM6001 ( n = 4 mice per group). (C) Measurement of soluble TREM2 (sTREM2) levels in the serum of NCD- or HFD-fed mice treated with GM6001 for 10 weeks ( n = 3 mice per group). (D–F) Representative flow profile and quantification of (D) CD11B + CD45 + cells (E) M2/M1 macrophage ratio (CD206 + CD11C − CD45 + CD11B + /CD11C + CD206 − CD45 + CD11B + ), and (F) TREM2 + CD11C + and TREM2 + CD206 + macrophage populations in GWAT of GM6001-treated mice after feeding HFD for 10 weeks ( n = 4 mice per group). (G) Immunofluorescence staining of F4/80 (green) with DAPI (blue) counterstaining in paraffin-embedded GWAT sections from GM6001-treated and control mice ( n = 4 mice per group, scale bars, 100 μm). (H) Immunoblot analysis of P-STING, STING, NLRP3, F4/80, and caspase-1 expression levels in GWAT of HFD-fed mice treated with or without GM6001 ( n = 4 mice per group). Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: ADAM10 (B-3) Mouse mAb , Santa Cruz Biotechnology , Cat# sc-28358; RRID: AB_626636.

Techniques: Western Blot, Immunofluorescence, Staining, Control, Expressing

Journal: Nucleic Acids Research

Article Title: Selenite targets eIF4E-binding protein-1 to inhibit translation initiation and induce the assembly of non-canonical stress granules

doi: 10.1093/nar/gks566

Figure Lengend Snippet: Accumulation of various canonical SG components in selenite-induced SGs. The accumulation of canonical SG components, including ( A ) RNA-binding proteins (Ago2, FMR1, HuR and PCBP2) and O-GlcNAc-modified proteins, ( B ) translation initiation factors other than the ones tested in (eIF5A and eIF3e) and the small ribosomal S14 protein (Rps14) and ( C ) SG constituents that do not possess obvious RNA-binding activity (Rsk2, RACK1, importin α1/β1 and HDAC6) were probed for SG localization by immunostaining. Arrows indicate localization to SGs, whereas arrowheads indicate the lack of enrichment. Bar = 10 µm.

Article Snippet: The following antibodies were used for western blotting and immunofluorescence: anti-eIF3b (goat polyclonal, N-20), eIF3e (goat polyclonal, C-20), eIF4E (mouse monoclonal, P-2), eIF4G1 (rabbit polyclonal, H-300), G3BP1 (mouse monoclonal, TT-Y), FMR1 (mouse monoclonal, 148.1), Hedls (mouse monoclonal, originally intended to react with S6K1 , H-9), HDAC6 (rabbit polyclonal, H-300), HuR (mouse monoclonal, 3A2), PABP (mouse monoclonal, 10E10), RACK1 (mouse monoclonal, B-3) and TIAR (goat polyclonal, C-18) from Santa Cruz Biotechnology; anti-eIF4E-binding protein1 (4EBP1) (rabbit polyclonal), phospho-4EBP1 (Thr37/46, rabbit monoclonal), eIF4A1 (rabbit monoclonal), ribosomal S6 (mouse monoclonal), phospho-ribosomal S6 (rabbit polyclonal) and Rsk2 (rabbit monoclonal) from Cell Signaling Technology; anti-β-actin (mouse monoclonal) from Chemicon; anti-eIF5A (mouse monoclonal) from BD Biosciences; anti-G3BP1 (rabbit polyclonal) from Bethyl; anti-phospho-eIF2α (rabbit polyclonal) from Assay Designs; anti-Ago2 (mouse monoclonal) from Wako Bioproducts; anti-PCBP2 (mouse monoclonal) from Abnova; anti-O-GlcNAc (mouse IgM, CTD110.6) from Covance; anti-importin β1 (mouse monoclonal) from Pierce; anti-small ribosomal S14 (rabbit polyclonal) from Proteintech group.

Techniques: RNA Binding Assay, Modification, Activity Assay, Immunostaining