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: Antibodies used in Western blotting were: rabbit polyclonal anti-human Glo1 (prepared in-house [ ]; rabbit polyclonal anti-human EIF2α (Cat no 9722), anti-human phospho-EIF2α [Ser51] (Cat no 9721) and anti-human beta-actin (Cat no 4967), rabbit monoclonal anti-human IRE1α, clone 14C10 (Cat no 3294), anti-human PERK, clone C33E10 (Cat no 3292), anti-human phospho-PERK [Thr980] (Cat no 3179) and anti-human TXNIP, clone D5F3E (Cat no 14715), and mouse monoclonal anti-human CHOP (Cat no 2895) purchased from Cell Signalling Technology (Danvers, MA, USA); rabbit monoclonal anti-human phospho-IRE1α [S724] (Cat no ab48187) and anti-human ATF6 (Cat no ab227830) purchased from Abcam (Cambridge, UK); rabbit polyclonal anti-human XBP1 (Cat no PA5-95260) purchased from ThermoFisher (Waltham, MA, USA); and goat polyclonal anti-rabbit IgG, HRP conjugate (Cat no 32260) and anti-mouse IgG, HRP conjugate (Cat no 31430) purchased from Invitrogen (Waltham, MA, USA).

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

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: Increased TXNIP and inflammatory signaling in high glucose concentration-induced ER stress in human aortal endothelial cells in vitro . Effect of XBP1 knockdown: a XBP1 mRNA, b MCP-1 mRNA, c IL-8 mRNA, and d TXNIP mRNA. Key: LG + NT, low glucose concentration + non-target siRNA; LG + XBP1KD, low glucose concentration + XBP1 siRNA; HG + NT, high glucose concentration + non-target siRNA; and HG + XBP1KD, high glucose concentration + XBP1 siRNA. Effect of miR-17 activity on inflammatory signalling: e Effect of miR-17 mimic on TXNIP mRNA; and f miR-17 inhibitor on TXNIP mRNA. Comparison of UPR-linked gene expression induced by high glucose concentration and treatment with Tunicamycin: g TXNIP mRNA, h GRP79 mRNA, and i CHOP mRNA at 24 h and 72 h, as indicated. Key: LG, low glucose concentration control; LG + NT, low glucose concentration + non-target siRNA control; LG + XBP1KD, low glucose concentration + XBP1 siRNA (knockdown); LG + miR17 m or miR17In, low glucose concentration + miR-17 mimic or inhibitor; HG, high glucose concentration; HG + NT, high glucose concentration + non-target siRNA; HG + XBP1KD, high glucose concentration + XBP1 siRNA (knockdown); and HG + miR17 m or miR17In, high glucose concentration + miR-17 mimic or 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 + XBP1KD, LG + miR17 m or LG + miR17In; and †† and †††, p < 0.01 and p < 0.001 with respect to HG or HG + NT control ( Student's t-test ). ANOVA : all P < 0.001. Incubations were for 72 h. Key to bar shading: solid pastel blue and red bars, low and high glucose concentration controls; pastel blue and red bars hatched bars, low and high glucose concentration with further additions, respectively; and grey bar, + Tunicamycin. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Antibodies used in Western blotting were: rabbit polyclonal anti-human Glo1 (prepared in-house [ ]; rabbit polyclonal anti-human EIF2α (Cat no 9722), anti-human phospho-EIF2α [Ser51] (Cat no 9721) and anti-human beta-actin (Cat no 4967), rabbit monoclonal anti-human IRE1α, clone 14C10 (Cat no 3294), anti-human PERK, clone C33E10 (Cat no 3292), anti-human phospho-PERK [Thr980] (Cat no 3179) and anti-human TXNIP, clone D5F3E (Cat no 14715), and mouse monoclonal anti-human CHOP (Cat no 2895) purchased from Cell Signalling Technology (Danvers, MA, USA); rabbit monoclonal anti-human phospho-IRE1α [S724] (Cat no ab48187) and anti-human ATF6 (Cat no ab227830) purchased from Abcam (Cambridge, UK); rabbit polyclonal anti-human XBP1 (Cat no PA5-95260) purchased from ThermoFisher (Waltham, MA, USA); and goat polyclonal anti-rabbit IgG, HRP conjugate (Cat no 32260) and anti-mouse IgG, HRP conjugate (Cat no 31430) purchased from Invitrogen (Waltham, MA, USA).

Techniques: Concentration Assay, In Vitro, Activity Assay, Comparison, Expressing

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: Antibodies used in Western blotting were: rabbit polyclonal anti-human Glo1 (prepared in-house [ ]; rabbit polyclonal anti-human EIF2α (Cat no 9722), anti-human phospho-EIF2α [Ser51] (Cat no 9721) and anti-human beta-actin (Cat no 4967), rabbit monoclonal anti-human IRE1α, clone 14C10 (Cat no 3294), anti-human PERK, clone C33E10 (Cat no 3292), anti-human phospho-PERK [Thr980] (Cat no 3179) and anti-human TXNIP, clone D5F3E (Cat no 14715), and mouse monoclonal anti-human CHOP (Cat no 2895) purchased from Cell Signalling Technology (Danvers, MA, USA); rabbit monoclonal anti-human phospho-IRE1α [S724] (Cat no ab48187) and anti-human ATF6 (Cat no ab227830) purchased from Abcam (Cambridge, UK); rabbit polyclonal anti-human XBP1 (Cat no PA5-95260) purchased from ThermoFisher (Waltham, MA, USA); and goat polyclonal anti-rabbit IgG, HRP conjugate (Cat no 32260) and anti-mouse IgG, HRP conjugate (Cat no 31430) purchased from Invitrogen (Waltham, MA, USA).

Techniques: Modification, Activation Assay, Derivative Assay, Binding Assay, Expressing