Journal: Redox Biology

Article Title: Increased cellular protein modification by methylglyoxal activates endoplasmic reticulum-based sensors of the unfolded protein response

doi: 10.1016/j.redox.2024.103025

Figure Lengend Snippet: Effect of dicarbonyl stress and high glucose concentration on activation of the IRE1α sensor pathway of the UPR in human aortal endothelial cells in vitro . Effect of Glo1 silencing – Western blotting: a Glo1, b pIRE1α, c total IRE1α, d pIRE1α/total IRE1α ratio (ratio of bands in b and c ), e XBP1s, f XBP1u, g XBP1s/XBP1u ratio (ratio of bands in e and f ), and h TXNIP. Key: LG + NT, low glucose concentration (4.1 mM) + non-target siRNA; LG + Glo1KD, low glucose concentration + Glo1 siRNA (knockdown); HG + NT, high glucose concentration (20 mM) + non-target siRNA; and HG + Glo1KD, high glucose concentration + Glo1 siRNA. i and j Effect of IRE1α inhibitor, 4μ8C, on XBP1 mRNA splicing and expression of TXNIP, respectively. Effect of miR-17 agonism and inhibition on expression of TXNIP: k TXNIP mRNA with and without miR-17 mimic (miR17m); and l , TXNIP mRNA with and without miR-17 inhibitor (miR17In). Key: LG, low glucose concentration (4.1 mM); LG+4μ8C/miR17m/miR17In, low glucose concentration + 4μ8C, miR-17 mimic or miR-17 inhibitor; HG, high glucose concentration; HG+4μ8C/miR17m/miR17In, high glucose concentration + 4μ8C, miR-17 mimic or miR-17 inhibitor. Data are mean ± SD (n = 3). Significance: *, ** and ***, p < 0.05, p < 0.01 and p < 0.001 with respect to LG or LG + NT control; o, oo and ooo, p < 0.05, p < 0.01 and p < 0.001 with respect to LG + Glo1KD, 4μ8C, miR-17 m or miR-17In; and †, †† and †††, p < 0.05, p < 0.01 and p < 0.001 with respect to HG or HG + NT control ( Student's t-test ). ANOVA : p < 0.001 except for c (P < 0.05) and f (P < 0.01). Incubations were for 72 h. Key to bar shading: solid pastel blue and red bars, low and high glucose concentration controls, respectively; pastel blue and red bars hatched bars, low and high glucose concentration with further additions, respectively; and grey bars in i , XBP1s mRNA. Abbreviation: ACTB, β-actin housekeeping protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Chemicals used were: IRE1α inhibitor, 7-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-8-carboxaldehyde 4μ8C and hesperetin, purchased from Merck, Darmstadt, Germany (Cat no SML-0949 and W431300, respectively); tunicamycin, purchased from Cell Signalling Biotechnology (Cat no 12819S); LipofectamineTM RNAiMAX Transfection Reagent, purchased from ThermoFisher (cat no 13778500); and tran s-resveratrol, purchased from Cayman Chemical Company, Ann Arbor, MI, USA (Cat no 70675).

Techniques: Concentration Assay, Activation Assay, In Vitro, Western Blot, Knockdown, Expressing, Inhibition, Control

Journal: Redox Biology

Article Title: Increased cellular protein modification by methylglyoxal activates endoplasmic reticulum-based sensors of the unfolded protein response

doi: 10.1016/j.redox.2024.103025

Figure Lengend Snippet: Effect of glyoxalase 1 overexpression and induction by Glo1 inducer, trans -resveratrol and hesperetin, on activation of IRE1α and signalling in human endothelial cells in vitro . a - g Effect of overexpression of Glo1 on activation of the UPR. Western blotting: a Glo1, b pIRE1α, c TXNIP, d XBP1u, e XBP1s, f ATF6 and g ATF6-N. h – k Effect of Glo1 inducer, tRES + HESP, on activation of the UPR. Western blotting: h pIRE1α, i XBP1s, j TXNIP, and k CHOP protein. Key: LG, low glucose concentration; LG + EV, low glucose concentration + empty vector; LG + Glo1, low glucose concentration + Glo1 overexpression vector; LG + RH, low glucose concentration +5 μM tRES + HESP; HG, high glucose concentration; HG + EV, high glucose concentration + empty vector; HG + Glo1, high glucose concentration + Glo1 overexpression vector; and HG + RH, high glucose concentration +5 μM tRES + HESP. Data are mean ± SD (n = 3). Significance: *, ** and ***, p < 0.05, p < 0.01 and p < 0.001 with respect to LG + EV or LG control; o, oo and ooo, p < 0.05, p < 0.01 and p < 0.001 with respect to HG + EV or HG control; and †, ††, and †††, p < 0.05, p < 0.01 and p < 0.001 with respect to HG or HG + EV control; and o and oo, p < 0.05 and p < 0.01 with respect to LG + Glo1 or LG + RH control; ( Student's t-test ). ANOVA : P < 0.001 except h , p < 0.01. Incubations were for 72 h. Key to bar shading: solid pastel blue and red bars, low and high glucose concentration controls, respectively, with or without empty vector transfection; and pastel blue and red bars hatched bars, low and high glucose concentration, respectively, with further additions (Glo1 overexpression vector or 5 μM tRES + HESP), respectively. Abbreviation: ACTB, β-actin housekeeping protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Chemicals used were: IRE1α inhibitor, 7-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-8-carboxaldehyde 4μ8C and hesperetin, purchased from Merck, Darmstadt, Germany (Cat no SML-0949 and W431300, respectively); tunicamycin, purchased from Cell Signalling Biotechnology (Cat no 12819S); LipofectamineTM RNAiMAX Transfection Reagent, purchased from ThermoFisher (cat no 13778500); and tran s-resveratrol, purchased from Cayman Chemical Company, Ann Arbor, MI, USA (Cat no 70675).

Techniques: Over Expression, Activation Assay, In Vitro, Western Blot, Concentration Assay, Plasmid Preparation, Control, Transfection

Journal: Redox Biology

Article Title: Increased cellular protein modification by methylglyoxal activates endoplasmic reticulum-based sensors of the unfolded protein response

doi: 10.1016/j.redox.2024.103025

Figure Lengend Snippet: Pathways of the UPR – indicating interactions with methylglyoxal and methylglyoxal-modified proteins. Blue arrows are processes of UPR sensor activation and deactivation; yellow arrows are UPR signalling; and red arrows are PDI modification by MG. Abbreviations: ATF3, ATF4, ATF5 and ATF6, activating transcription factor-3, -4, -5 and -6; BH3, proteins with 3 domains homologous to BCL-2; CHOP, C/EBP homologous protein; Dr5, death receptor-5; eIF2α, eukaryotic translation initiation factor-2α; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated protein degradation; Gadd34, growth arrest and DNA damage-inducible protein; GRP78, 78 kDa glucose-regulated protein; ICAM-1, intercellular adhesion molecule-1; IL-1β, −6, −8 and −18; interleukin-1β, −6, −8 and −18; IRE1α, inositol requiring enzyme-1α; MCP-1, monocyte chemoattractant-1; MG, methylglyoxal; MG-H1, methylglyoxal-derived hydroimidazolone; miR-17, microRNA-17; NLRP3, nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3; P, protein phosphorylation; PDI, protein disulfide isomerase; PERK, double-stranded RNA-dependent kinase-like ER kinase; RIDD, regulated IRE1α-dependent decay; S1P/S2P, site-1 protease/site-2 protease; TNFα, tumor necrosis factor-α; TXNIP, thioredoxin interacting protein; VCAM-1, vascular cell adhesion molecule-1; XBP1, X-box binding protein 1 (subscripts u and s indicate unprocessed mRNA and spliced mRNA expression products, respectively). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Chemicals used were: IRE1α inhibitor, 7-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-8-carboxaldehyde 4μ8C and hesperetin, purchased from Merck, Darmstadt, Germany (Cat no SML-0949 and W431300, respectively); tunicamycin, purchased from Cell Signalling Biotechnology (Cat no 12819S); LipofectamineTM RNAiMAX Transfection Reagent, purchased from ThermoFisher (cat no 13778500); and tran s-resveratrol, purchased from Cayman Chemical Company, Ann Arbor, MI, USA (Cat no 70675).

Techniques: Modification, Activation Assay, Derivative Assay, Binding Assay, Phospho-proteomics, Expressing