Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD binds to GSTM2 and stabilize its protein levels (A) Three-dimensional schematic diagram of the interaction between HCY-NBD and GSTM2. The yellow and gray structures represent the 3D conformation of GSTM2 protein, while HCY-NBD is depicted as a green stick model. Molecular docking simulations predict binding affinity between GSTM2 and HCY-NBD with a binding energy of −7.52 kcal/mol. (B) Two-dimensional interaction diagram between HCY-NBD and GSTM2. Green dashed lines indicate hydrogen bonds, light green dashed lines represent carbon-hydrogen bonds, and pink dashed lines denote hydrophobic interactions. (C) Binding kinetics between HCY-NBD and GSTM2 measured by Biacore T200. (D) GSTM2 enzymatic activity kinetics was measured in vitro following exposure to either DMSO solvent control or HCY-NBD. GS-DNB refers to the product of the GSTM2 enzymatic activity assay. (E) HUVEC cells were treated with increasing glucose concentrations (0, 10, 30, 60 mM) for 24 h, followed by quantification of GSTM2 mRNA levels via qPCR. (F) HUVECs were treated with HCY-NBD (0, 1, 3, 5 μM) for 24 h under HG (30 mM) conditions, the GSTM2 mRNA expression were analyzed by qPCR. (G – H) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the GSTM2 protein levels were determined by Western blot. (I – J) HUVECs were treated with HCY-NBD (0, 1, 3, 5 μM) for 24 h under HG (30 mM) conditions, the GSTM2 protein levels were determined by Western blot. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: Briefly, 2 μg of GSTM2 (HY–P75152A, MCE) protein was incubated with DMSO or HCY-NBD (0.1, 0.5, 1, 3, 5 μM) on ice for 1 h. The enzymatic reaction was initiated by adding the reaction mixture containing 1 mM GSH and 1 mM CDNB in a 96-well UV-transparent microplate.

Techniques: Binding Assay, Activity Assay, In Vitro, Solvent, Control, Enzyme Activity Assay, Expressing, Western Blot

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD up-regulates GSTM2 sulfenylation and inhibits GSTM2 ubiquitination (A-B) HUVECs were pretreated with 80 μM HDX for 1h, then the cells were treated with HCY-NBD (0 or 5 μM) and HDX (0 or 80 μM) under HG (30 mM) conditions for 24 h. Protein levels of GSTM2 were analyzed by Western blot. (C – D) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the sulfenylation levels of GSTM2 were measured by Western blot. (E – F) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the ubiquitination levels of GSTM2 were determined by Western blot. (G – H) HUVECs were transfected with GFP-GSTM2, and HA-Ub (Wild-type, K48-only, or K63-only), exposed to designated treatments, and assessed for GFP-GSTM2 ubiquitination levels via Western blot in all experimental groups. (I – L) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the glutathionylation levels of GSTM2 were determined by Western blot. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: Briefly, 2 μg of GSTM2 (HY–P75152A, MCE) protein was incubated with DMSO or HCY-NBD (0.1, 0.5, 1, 3, 5 μM) on ice for 1 h. The enzymatic reaction was initiated by adding the reaction mixture containing 1 mM GSH and 1 mM CDNB in a 96-well UV-transparent microplate.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: Cys174 is a critical residue for HCY-NBD-mediated regulation of GSTM2 sulfenylation and non-lysine ubiquitination (A-B) HUVECs were transfected with wide type (WT) and four distinct cysteine-to-alanine mutants (C87A, C115A, C174A, and C87-115-174A) of GSTM2, exposed to designated treatments, and assessed for GSTM2 sulfenylation levels via Western blot in all experimental groups. (C) HEK-293T cells were transfected with a GSTM2-overexpressing plasmid for 48 h. Ubiquitination at Cys174 was detected via LC-MS/MS, with MS/MS analysis demonstrating a characteristic +114.043 Da mass shift (C [114.043]). Score, 131.0. (D – E) HUVECs were transfected with wide type (WT) and 174 cysteine-to-alanine(C174A) mutants of GSTM2, exposed to designated treatments, and assessed for GSTM2 ubiquitination levels via Western blot in all experimental groups. (F – G) HUVECs were transfected with GFP-GSTM2-WT, GFP-GSTM2-C174A, and HA-Ub (WT, K48-only), exposed to designated treatments, and assessed for GFP-GSTM2 ubiquitination levels via Western blot in all experimental groups. (H–K) HUVECs were transfected with GFP-GSTM2-WT or GFP-GSTM2-C174A 24 h, then treated with cycloheximide (2.5 μM) and designated treatments for the indicated times, followed by Western blotting. (L – M) HUVECs were transfected with wide type (WT) and 174 cysteine-to-alanine(C174A) mutants of GSTM2, exposed to designated treatments, and assessed for HA levels via Western blot in all experimental groups. H–K: Two-way ANOVA was performed, others: one-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: Briefly, 2 μg of GSTM2 (HY–P75152A, MCE) protein was incubated with DMSO or HCY-NBD (0.1, 0.5, 1, 3, 5 μM) on ice for 1 h. The enzymatic reaction was initiated by adding the reaction mixture containing 1 mM GSH and 1 mM CDNB in a 96-well UV-transparent microplate.

Techniques: Residue, Ubiquitin Proteomics, Transfection, Western Blot, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD inhibits high glucose-induced senescence of HUVECs (A-D) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the protein levels of p21, p16 and p53 were determined by Western blot. (E – F) Cell cycle analysis by flow cytometry for HUVECs in different groups. (G – H) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, SA- β -gal staining was performed to observe senescence in HUVECs. (I – L) HUVECs were transfected with GSTM2 siRNA at a final concentration of 50 nM, while control groups were transfected with scramble siRNA at the same concentration. After 24 h, cells cultured under a HG (30 mM) model were treated with 0 or 5 μM HCY-NBD for 24 h. Protein levels of p21 were analyzed by Western blot and SA- β -gal staining was performed to observe senescence in HUVECs. (M – N) HUVECs were transfected with wide type (WT) and four distinct cysteine-to-alanine mutants (C87A, C115A, C174A, and C87-115-174A) of GSTM2, exposed to designated treatments, and assessed for protein levels of p21 via Western blot in all experimental groups. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3, n = 4(E–F).

Article Snippet: Briefly, 2 μg of GSTM2 (HY–P75152A, MCE) protein was incubated with DMSO or HCY-NBD (0.1, 0.5, 1, 3, 5 μM) on ice for 1 h. The enzymatic reaction was initiated by adding the reaction mixture containing 1 mM GSH and 1 mM CDNB in a 96-well UV-transparent microplate.

Techniques: Western Blot, Cell Cycle Assay, Flow Cytometry, Staining, Transfection, Concentration Assay, Control, Cell Culture

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD inhibits vascular senescence in db/db mice (A) Experimental Workflow Diagram. (B) HCY-NBD was administered intraperitoneally to db/db mice (0, 1, or 5 mg/kg per injection, every other day) over an 8-week period. Body weights were recorded weekly, n = 5. (C–H) Following 8 weeks of treatment, mice were humanely euthanized. Major organs (heart, liver, spleen, lungs, and kidneys) were excised and weighed using an analytical balance. Bar charts depict organ-to-body weight ratios, calculated as (organ weight/total body weight) × 100 %, n = 5. (I) Histopathological evaluation of heart, liver, spleen, lung, and kidney tissues was performed on H&E-stained sections to characterize microstructural organization and injury phenotypes, n = 4. (J – L) Thoracic aortic segments were subjected to en face immunofluorescence staining to assess p21 levels within CD31-positive endothelial cells and CD31 levels. Nuclei were counterstained with DAPI. n = 4. (M – N) Vascular senescence in thoracic aortas was detected using SA- β -gal staining, n = 3. (O–P) Protein expression levels of GSTM2 in thoracic aortas of db/db mice were detected via immunohistochemistry, n = 5. (Q – R) Immunofluorescence analysis detected protein expression levels of GSTM2 colocalized with CD31 in thoracic aortas of db/db mice, n = 4. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Briefly, 2 μg of GSTM2 (HY–P75152A, MCE) protein was incubated with DMSO or HCY-NBD (0.1, 0.5, 1, 3, 5 μM) on ice for 1 h. The enzymatic reaction was initiated by adding the reaction mixture containing 1 mM GSH and 1 mM CDNB in a 96-well UV-transparent microplate.

Techniques: Injection, Mass Measurement, Staining, Immunofluorescence, Expressing, Immunohistochemistry

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: Schematic presentation of the new approach to enhance endogenous GSTM2 level that inhibits high glucose-induced senescence and calcification in vascular endothelial cells.

Article Snippet: Briefly, 2 μg of GSTM2 (HY–P75152A, MCE) protein was incubated with DMSO or HCY-NBD (0.1, 0.5, 1, 3, 5 μM) on ice for 1 h. The enzymatic reaction was initiated by adding the reaction mixture containing 1 mM GSH and 1 mM CDNB in a 96-well UV-transparent microplate.

Techniques:

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM3P1/Gstm2-ps1 are transiently induced in kidney proximal tubular cells in sepsis-associated acute kidney injury models. BUMPT and HK-2 cells were treated with 100 μg/mL lipopolysaccharide (LPS) for 3, 6, and 12 hours. Male C57BL/6J mice were subjected to an i.p. injection of 10 mg/kg LPS or cecal ligation and puncture (CLP) surgery, and kidney samples were collected at 3, 6, and 9 hours after injury. A—D: Quantitative RT-PCR analysis of Gstm2-ps1 or GSTM3P1 expression. A: Gstm2-ps1 in BUMPT. B: GSTM3P1 in HK2. C: Gstm2-ps1 in LPS-treated mouse kidney. D: Gstm2-ps1 in mouse kidney with CLP. A—D: The statistical difference was determined using one-way analysis of variance with Tukey multiple comparison. E and F: Representative images of Gstm2-ps1 in kidney tissue assessed by in situ hybridization and costained with fluorescein isothiocyanate—labeled Lotus tetragonolobus lectin (LTL). E: Mouse kidneys with or without LPS treatment for 3 hours. F: Mouse kidneys with or without CLP injury for 3 hours. n 5 ( A—D ); n 6 ( E and F ). ** P < 0.01, *** P < 0.001. Scale bar 100 μm ( E and F ). NS, nonsignificant.

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Injection, Ligation, Quantitative RT-PCR, Expressing, Comparison, In Situ Hybridization, Labeling

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM3P1/Gstm2-ps1 exacerbate lipopolysaccharide (LPS)—induced apoptosis in cultured proximal tubular cells. RPTC cells with or without GSTM3P1 stable overexpression, BUMPT cells with or without Gstm2-ps1 stable overexpression, and wild-type (WT) or Gstm2-ps1 knockout (KO) primary proximal tubular cells were treated with 100 μg/mL LPS for 19 hours. A—C: Quantitative RT-PCR analysis confirming the expression of GSTM3P1 or Gstm2-ps1. A: RPTC. B: BUMPT. C: Primary proximal tubular cells. D—F: Cell viability assessed by MTT assay following 19 hours of LPS treatment. D: RPTC. E: BUMPT. F: Primary proximal tubular cells. A—F: The statistical difference was determined by unpaired t -test. G—I: Representative immunoblots of full-length caspase 3 and cleaved caspase 3 (C-caspase-3) with β-actin as the loading control (Ctrl; top panels ), and the densitometry analysis of C-caspase-3 normalized to β-actin ( bottom panels ). G: RPTC. H: BUMPT. I: Primary proximal tubular cells. G—I: The statistical difference was determined by two-way analysis of variance with Tukey multiple comparison. n =3 ( A , G , and H ); n = 4 ( B , C , and I ); n = 6 ( D—F ). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Cell Culture, Over Expression, Knock-Out, Quantitative RT-PCR, Expressing, MTT Assay, Western Blot, Control, Comparison

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM3P1/Gstm2-ps1 overexpression induces oxidative stress in cultured proximal tubular cells. RPTC cells with or without GSTM3P1 stable overexpression and BUMPT cells with or without Gstm2-ps1 stable overexpression were treated with 100 μg/mL lipopolysaccharide (LPS) for 19 hours. A and B: Oxidative stress levels were assessed by measuring the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio. A: RPTC. B: BUMPT. The statistical difference was determined by two-way analysis of variance with Tukey multiple comparison. C: Representative images of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA)—stained RPTC and phase-contrast images. n = 6 ( A—C ). * P < 0.05, ** P < 0.01, and *** P < 0.001. Scale bars = 200 μm ( C ).

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Over Expression, Cell Culture, Comparison, Staining

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: Proximal tubular—specific Gstm2-ps1 knockout protects mice from lipopolysaccharide (LPS)—induced sepsis acute kidney injury. Proximal tubular—specific Gstm2-ps1 knockout (KO) mice and their wild-type (WT) littermates were treated with one i.p. injection of 10 mg/kg LPS and sacrificed after 19 hours. The control (Ctrl) mice were given an equal volume of phosphate-buffered saline. A: Blood urea nitrogen (BUN) levels. B: Serum creatinine levels. C: Quantification of apoptotic cells in kidneys by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. A—C: The statistical difference was determined by two-way analysis of variance with Tukey multiple comparison. D: Representative images of TUNEL staining. E: Representative images of neutrophil gelatinase-associated lipocalin (NGAL) immunohistochemical staining. F: Representative images of reactive oxygen species (ROS) levels detected by 2′,7′-dichlorodihydrofluorescein diacetate staining. G: Representative immunoblots of kidney injury molecule-1 (KIM-1) and the densitometry analysis of KIM-1 normalized to the internal loading control glyceraldehyde-3-phosphate dehydrogenase. Data are presented as means ± SD ( A—C and G ). n = 6 per group ( A , B , E , and F ); n = 5 per group ( C ); n = 4 per group ( G ). ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Scale bars: 50 μm ( D and E ); 100 μm ( F ).

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Knock-Out, Injection, Control, Saline, End Labeling, TUNEL Assay, Staining, Comparison, Immunohistochemical staining, Western Blot

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM3P1/Gstm2-ps1 down-regulate the expression of their parent gene GSTM3/GSTM2. A—F: RPTC cells ( A and D ) or BUMPT cells ( B and E ) were overexpressed with or without GSTM3P1 or Gstm2-ps1, and C57BL/6J mouse kidneys ( C and F ) were treated with 10 mg/kg lipopolysaccharide (LPS) for 3, 6, and 9 hours. A—C: Representative immunoblots of GSTM3 and GSTM2, with β-actin or cyclophilin B as the internal loading marker. D—F: The densitometric analysis of GSTM3 and GSTM2 protein normalized to the internal loading control. The statistical difference was determined by unpaired t -test ( D and E ) or one-way analysis of variance with Tukey multiple comparison ( F ). G: Representative immunofluorescence images of GSTM2 with fluorescein isothiocyanate— Lotus tetragonolobus lectin (LTL) costaining. Gstm2-ps1 knockout (KO) and wild-type (WT) mice were treated with/without 10 mg/kg LPS for 3 hours. n = 6 for cell samples ( D—F ); n = 4 for kidney samples ( D—F ); n = 6 ( G ). ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Scale bar = 100 μm ( G ). NS, nonsignificant.

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Expressing, Western Blot, Marker, Control, Comparison, Immunofluorescence, Knock-Out

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM2/GSTM3 protect cultured renal proximal tubular cell from lipopolysaccharide (LPS)—induced apoptosis. RPTC cells with or without GSTM3 overexpression, and BUMPT cells with or without GSTM2 overexpression, were treated with 100 μg/mL LPS. A and B: Immunoblot analysis confirming the overexpression efficiency of GSTM3 and GSTM2. C and D: Cell viability assessed using the MTT assay following 17 hours of LPS treatment. A—D: The statistical difference was determined by unpaired t -test. E and F: Representative immunoblots ( top panels ) and densitometric analysis ( bottom panels ) of cleaved caspase-3 (C-caspase-3) protein levels. E: RPTC. F: BUMPT. E and F: The statistical difference was determined by one-way analysis of variance with Tukey multiple comparison. n = 3 ( A and B ); n = 6 ( C and D ); n = 4 ( E and F ). ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Ctrl, control.

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Cell Culture, Over Expression, Western Blot, MTT Assay, Comparison, Control

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM3P1/Gstm2-ps1 may down-regulate GSTM3/GSTM2 protein level through RNA translation inhibition. A: Quantitative RT-PCR (RT-qPCR) of GSTM3 in RPTC cells with or without GSTM3P1 overexpression. The unpaired t -test was used. B: RT-qPCR of GSTM2 in BUMPT cells with or without Gstm2-ps1 overexpression. The unpaired t -test was used. C: RT-qPCR of GSTM2 in mouse kidneys with or without 10 mg/kg lipopolysaccharide (LPS) treatment . The one-way analysis of variance (ANOVA) with Tukey multiple comparison was used. D and E: BUMPT cell lysates with Gstm2-ps1 overexpression or empty vector transfection (pcDNA) were subjected to RNA pull-down assay with Gstm2-ps1 probes. The input lysates (Input) and pull-down products (pull-down) were examined by immunoblotting of human antigen R (HuR), with cyclophilin B as the internal loading control of input lysates. D: Representative immunoblots of HuR and cyclophilin B. E: The densitometry analysis of HuR immunoblotting in pull-down products normalized by its level in the input. The unpaired t -test was used. F—H: BUMPT cells were overexpressed with HuR or Gstm2-ps1. F: Representative immunoblots to confirm the overexpression of HuR with cyclophilin B as the internal loading control. The upper band is the overexpressed HuR. The lower band is the endogenous HuR. G: Representative immunoblots and densitometry analysis of GSTM2 in cells with or without Gstm2-ps1 and HuR overexpression. β-Actin was used as the internal loading control. The one-way ANOVA with Tukey multiple comparison was used. H: The cell viability assessed by MTT assay. The one-way ANOVA with Tukey multiple comparison was used. n = 5 ( A and B ); n = 4 ( C and E—G ); n = 6 ( H ). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. NS, nonsignificant.

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Inhibition, Quantitative RT-PCR, Over Expression, Comparison, Plasmid Preparation, Transfection, Pull Down Assay, Western Blot, Control, MTT Assay

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD binds to GSTM2 and stabilize its protein levels (A) Three-dimensional schematic diagram of the interaction between HCY-NBD and GSTM2. The yellow and gray structures represent the 3D conformation of GSTM2 protein, while HCY-NBD is depicted as a green stick model. Molecular docking simulations predict binding affinity between GSTM2 and HCY-NBD with a binding energy of −7.52 kcal/mol. (B) Two-dimensional interaction diagram between HCY-NBD and GSTM2. Green dashed lines indicate hydrogen bonds, light green dashed lines represent carbon-hydrogen bonds, and pink dashed lines denote hydrophobic interactions. (C) Binding kinetics between HCY-NBD and GSTM2 measured by Biacore T200. (D) GSTM2 enzymatic activity kinetics was measured in vitro following exposure to either DMSO solvent control or HCY-NBD. GS-DNB refers to the product of the GSTM2 enzymatic activity assay. (E) HUVEC cells were treated with increasing glucose concentrations (0, 10, 30, 60 mM) for 24 h, followed by quantification of GSTM2 mRNA levels via qPCR. (F) HUVECs were treated with HCY-NBD (0, 1, 3, 5 μM) for 24 h under HG (30 mM) conditions, the GSTM2 mRNA expression were analyzed by qPCR. (G – H) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the GSTM2 protein levels were determined by Western blot. (I – J) HUVECs were treated with HCY-NBD (0, 1, 3, 5 μM) for 24 h under HG (30 mM) conditions, the GSTM2 protein levels were determined by Western blot. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: GSTM2 (sc-376486) antibodies were acquired from Santa Cruz Biotechnology.

Techniques: Binding Assay, Activity Assay, In Vitro, Solvent, Control, Enzyme Activity Assay, Expressing, Western Blot

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD up-regulates GSTM2 sulfenylation and inhibits GSTM2 ubiquitination (A-B) HUVECs were pretreated with 80 μM HDX for 1h, then the cells were treated with HCY-NBD (0 or 5 μM) and HDX (0 or 80 μM) under HG (30 mM) conditions for 24 h. Protein levels of GSTM2 were analyzed by Western blot. (C – D) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the sulfenylation levels of GSTM2 were measured by Western blot. (E – F) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the ubiquitination levels of GSTM2 were determined by Western blot. (G – H) HUVECs were transfected with GFP-GSTM2, and HA-Ub (Wild-type, K48-only, or K63-only), exposed to designated treatments, and assessed for GFP-GSTM2 ubiquitination levels via Western blot in all experimental groups. (I – L) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the glutathionylation levels of GSTM2 were determined by Western blot. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: GSTM2 (sc-376486) antibodies were acquired from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: Cys174 is a critical residue for HCY-NBD-mediated regulation of GSTM2 sulfenylation and non-lysine ubiquitination (A-B) HUVECs were transfected with wide type (WT) and four distinct cysteine-to-alanine mutants (C87A, C115A, C174A, and C87-115-174A) of GSTM2, exposed to designated treatments, and assessed for GSTM2 sulfenylation levels via Western blot in all experimental groups. (C) HEK-293T cells were transfected with a GSTM2-overexpressing plasmid for 48 h. Ubiquitination at Cys174 was detected via LC-MS/MS, with MS/MS analysis demonstrating a characteristic +114.043 Da mass shift (C [114.043]). Score, 131.0. (D – E) HUVECs were transfected with wide type (WT) and 174 cysteine-to-alanine(C174A) mutants of GSTM2, exposed to designated treatments, and assessed for GSTM2 ubiquitination levels via Western blot in all experimental groups. (F – G) HUVECs were transfected with GFP-GSTM2-WT, GFP-GSTM2-C174A, and HA-Ub (WT, K48-only), exposed to designated treatments, and assessed for GFP-GSTM2 ubiquitination levels via Western blot in all experimental groups. (H–K) HUVECs were transfected with GFP-GSTM2-WT or GFP-GSTM2-C174A 24 h, then treated with cycloheximide (2.5 μM) and designated treatments for the indicated times, followed by Western blotting. (L – M) HUVECs were transfected with wide type (WT) and 174 cysteine-to-alanine(C174A) mutants of GSTM2, exposed to designated treatments, and assessed for HA levels via Western blot in all experimental groups. H–K: Two-way ANOVA was performed, others: one-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: GSTM2 (sc-376486) antibodies were acquired from Santa Cruz Biotechnology.

Techniques: Residue, Ubiquitin Proteomics, Transfection, Western Blot, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD inhibits high glucose-induced senescence of HUVECs (A-D) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the protein levels of p21, p16 and p53 were determined by Western blot. (E – F) Cell cycle analysis by flow cytometry for HUVECs in different groups. (G – H) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, SA- β -gal staining was performed to observe senescence in HUVECs. (I – L) HUVECs were transfected with GSTM2 siRNA at a final concentration of 50 nM, while control groups were transfected with scramble siRNA at the same concentration. After 24 h, cells cultured under a HG (30 mM) model were treated with 0 or 5 μM HCY-NBD for 24 h. Protein levels of p21 were analyzed by Western blot and SA- β -gal staining was performed to observe senescence in HUVECs. (M – N) HUVECs were transfected with wide type (WT) and four distinct cysteine-to-alanine mutants (C87A, C115A, C174A, and C87-115-174A) of GSTM2, exposed to designated treatments, and assessed for protein levels of p21 via Western blot in all experimental groups. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3, n = 4(E–F).

Article Snippet: GSTM2 (sc-376486) antibodies were acquired from Santa Cruz Biotechnology.

Techniques: Western Blot, Cell Cycle Assay, Flow Cytometry, Staining, Transfection, Concentration Assay, Control, Cell Culture

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD inhibits vascular senescence in db/db mice (A) Experimental Workflow Diagram. (B) HCY-NBD was administered intraperitoneally to db/db mice (0, 1, or 5 mg/kg per injection, every other day) over an 8-week period. Body weights were recorded weekly, n = 5. (C–H) Following 8 weeks of treatment, mice were humanely euthanized. Major organs (heart, liver, spleen, lungs, and kidneys) were excised and weighed using an analytical balance. Bar charts depict organ-to-body weight ratios, calculated as (organ weight/total body weight) × 100 %, n = 5. (I) Histopathological evaluation of heart, liver, spleen, lung, and kidney tissues was performed on H&E-stained sections to characterize microstructural organization and injury phenotypes, n = 4. (J – L) Thoracic aortic segments were subjected to en face immunofluorescence staining to assess p21 levels within CD31-positive endothelial cells and CD31 levels. Nuclei were counterstained with DAPI. n = 4. (M – N) Vascular senescence in thoracic aortas was detected using SA- β -gal staining, n = 3. (O–P) Protein expression levels of GSTM2 in thoracic aortas of db/db mice were detected via immunohistochemistry, n = 5. (Q – R) Immunofluorescence analysis detected protein expression levels of GSTM2 colocalized with CD31 in thoracic aortas of db/db mice, n = 4. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: GSTM2 (sc-376486) antibodies were acquired from Santa Cruz Biotechnology.

Techniques: Injection, Mass Measurement, Staining, Immunofluorescence, Expressing, Immunohistochemistry

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: Schematic presentation of the new approach to enhance endogenous GSTM2 level that inhibits high glucose-induced senescence and calcification in vascular endothelial cells.

Article Snippet: GSTM2 (sc-376486) antibodies were acquired from Santa Cruz Biotechnology.

Techniques:

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD binds to GSTM2 and stabilize its protein levels (A) Three-dimensional schematic diagram of the interaction between HCY-NBD and GSTM2. The yellow and gray structures represent the 3D conformation of GSTM2 protein, while HCY-NBD is depicted as a green stick model. Molecular docking simulations predict binding affinity between GSTM2 and HCY-NBD with a binding energy of −7.52 kcal/mol. (B) Two-dimensional interaction diagram between HCY-NBD and GSTM2. Green dashed lines indicate hydrogen bonds, light green dashed lines represent carbon-hydrogen bonds, and pink dashed lines denote hydrophobic interactions. (C) Binding kinetics between HCY-NBD and GSTM2 measured by Biacore T200. (D) GSTM2 enzymatic activity kinetics was measured in vitro following exposure to either DMSO solvent control or HCY-NBD. GS-DNB refers to the product of the GSTM2 enzymatic activity assay. (E) HUVEC cells were treated with increasing glucose concentrations (0, 10, 30, 60 mM) for 24 h, followed by quantification of GSTM2 mRNA levels via qPCR. (F) HUVECs were treated with HCY-NBD (0, 1, 3, 5 μM) for 24 h under HG (30 mM) conditions, the GSTM2 mRNA expression were analyzed by qPCR. (G – H) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the GSTM2 protein levels were determined by Western blot. (I – J) HUVECs were treated with HCY-NBD (0, 1, 3, 5 μM) for 24 h under HG (30 mM) conditions, the GSTM2 protein levels were determined by Western blot. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: Green dashed lines indicate hydrogen bonds, light green dashed lines represent carbon-hydrogen bonds, and pink dashed lines denote hydrophobic interactions. (C) Binding kinetics between HCY-NBD and GSTM2 measured by Biacore T200. (D) GSTM2 enzymatic activity kinetics was measured in vitro following exposure to either DMSO solvent control or HCY-NBD.

Techniques: Binding Assay, Activity Assay, In Vitro, Solvent, Control, Enzyme Activity Assay, Expressing, Western Blot

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD up-regulates GSTM2 sulfenylation and inhibits GSTM2 ubiquitination (A-B) HUVECs were pretreated with 80 μM HDX for 1h, then the cells were treated with HCY-NBD (0 or 5 μM) and HDX (0 or 80 μM) under HG (30 mM) conditions for 24 h. Protein levels of GSTM2 were analyzed by Western blot. (C – D) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the sulfenylation levels of GSTM2 were measured by Western blot. (E – F) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the ubiquitination levels of GSTM2 were determined by Western blot. (G – H) HUVECs were transfected with GFP-GSTM2, and HA-Ub (Wild-type, K48-only, or K63-only), exposed to designated treatments, and assessed for GFP-GSTM2 ubiquitination levels via Western blot in all experimental groups. (I – L) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the glutathionylation levels of GSTM2 were determined by Western blot. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: Green dashed lines indicate hydrogen bonds, light green dashed lines represent carbon-hydrogen bonds, and pink dashed lines denote hydrophobic interactions. (C) Binding kinetics between HCY-NBD and GSTM2 measured by Biacore T200. (D) GSTM2 enzymatic activity kinetics was measured in vitro following exposure to either DMSO solvent control or HCY-NBD.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: Cys174 is a critical residue for HCY-NBD-mediated regulation of GSTM2 sulfenylation and non-lysine ubiquitination (A-B) HUVECs were transfected with wide type (WT) and four distinct cysteine-to-alanine mutants (C87A, C115A, C174A, and C87-115-174A) of GSTM2, exposed to designated treatments, and assessed for GSTM2 sulfenylation levels via Western blot in all experimental groups. (C) HEK-293T cells were transfected with a GSTM2-overexpressing plasmid for 48 h. Ubiquitination at Cys174 was detected via LC-MS/MS, with MS/MS analysis demonstrating a characteristic +114.043 Da mass shift (C [114.043]). Score, 131.0. (D – E) HUVECs were transfected with wide type (WT) and 174 cysteine-to-alanine(C174A) mutants of GSTM2, exposed to designated treatments, and assessed for GSTM2 ubiquitination levels via Western blot in all experimental groups. (F – G) HUVECs were transfected with GFP-GSTM2-WT, GFP-GSTM2-C174A, and HA-Ub (WT, K48-only), exposed to designated treatments, and assessed for GFP-GSTM2 ubiquitination levels via Western blot in all experimental groups. (H–K) HUVECs were transfected with GFP-GSTM2-WT or GFP-GSTM2-C174A 24 h, then treated with cycloheximide (2.5 μM) and designated treatments for the indicated times, followed by Western blotting. (L – M) HUVECs were transfected with wide type (WT) and 174 cysteine-to-alanine(C174A) mutants of GSTM2, exposed to designated treatments, and assessed for HA levels via Western blot in all experimental groups. H–K: Two-way ANOVA was performed, others: one-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: Green dashed lines indicate hydrogen bonds, light green dashed lines represent carbon-hydrogen bonds, and pink dashed lines denote hydrophobic interactions. (C) Binding kinetics between HCY-NBD and GSTM2 measured by Biacore T200. (D) GSTM2 enzymatic activity kinetics was measured in vitro following exposure to either DMSO solvent control or HCY-NBD.

Techniques: Residue, Ubiquitin Proteomics, Transfection, Western Blot, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD inhibits high glucose-induced senescence of HUVECs (A-D) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the protein levels of p21, p16 and p53 were determined by Western blot. (E – F) Cell cycle analysis by flow cytometry for HUVECs in different groups. (G – H) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, SA- β -gal staining was performed to observe senescence in HUVECs. (I – L) HUVECs were transfected with GSTM2 siRNA at a final concentration of 50 nM, while control groups were transfected with scramble siRNA at the same concentration. After 24 h, cells cultured under a HG (30 mM) model were treated with 0 or 5 μM HCY-NBD for 24 h. Protein levels of p21 were analyzed by Western blot and SA- β -gal staining was performed to observe senescence in HUVECs. (M – N) HUVECs were transfected with wide type (WT) and four distinct cysteine-to-alanine mutants (C87A, C115A, C174A, and C87-115-174A) of GSTM2, exposed to designated treatments, and assessed for protein levels of p21 via Western blot in all experimental groups. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3, n = 4(E–F).

Article Snippet: Green dashed lines indicate hydrogen bonds, light green dashed lines represent carbon-hydrogen bonds, and pink dashed lines denote hydrophobic interactions. (C) Binding kinetics between HCY-NBD and GSTM2 measured by Biacore T200. (D) GSTM2 enzymatic activity kinetics was measured in vitro following exposure to either DMSO solvent control or HCY-NBD.

Techniques: Western Blot, Cell Cycle Assay, Flow Cytometry, Staining, Transfection, Concentration Assay, Control, Cell Culture

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD inhibits vascular senescence in db/db mice (A) Experimental Workflow Diagram. (B) HCY-NBD was administered intraperitoneally to db/db mice (0, 1, or 5 mg/kg per injection, every other day) over an 8-week period. Body weights were recorded weekly, n = 5. (C–H) Following 8 weeks of treatment, mice were humanely euthanized. Major organs (heart, liver, spleen, lungs, and kidneys) were excised and weighed using an analytical balance. Bar charts depict organ-to-body weight ratios, calculated as (organ weight/total body weight) × 100 %, n = 5. (I) Histopathological evaluation of heart, liver, spleen, lung, and kidney tissues was performed on H&E-stained sections to characterize microstructural organization and injury phenotypes, n = 4. (J – L) Thoracic aortic segments were subjected to en face immunofluorescence staining to assess p21 levels within CD31-positive endothelial cells and CD31 levels. Nuclei were counterstained with DAPI. n = 4. (M – N) Vascular senescence in thoracic aortas was detected using SA- β -gal staining, n = 3. (O–P) Protein expression levels of GSTM2 in thoracic aortas of db/db mice were detected via immunohistochemistry, n = 5. (Q – R) Immunofluorescence analysis detected protein expression levels of GSTM2 colocalized with CD31 in thoracic aortas of db/db mice, n = 4. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Green dashed lines indicate hydrogen bonds, light green dashed lines represent carbon-hydrogen bonds, and pink dashed lines denote hydrophobic interactions. (C) Binding kinetics between HCY-NBD and GSTM2 measured by Biacore T200. (D) GSTM2 enzymatic activity kinetics was measured in vitro following exposure to either DMSO solvent control or HCY-NBD.

Techniques: Injection, Mass Measurement, Staining, Immunofluorescence, Expressing, Immunohistochemistry

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: Schematic presentation of the new approach to enhance endogenous GSTM2 level that inhibits high glucose-induced senescence and calcification in vascular endothelial cells.

Article Snippet: Green dashed lines indicate hydrogen bonds, light green dashed lines represent carbon-hydrogen bonds, and pink dashed lines denote hydrophobic interactions. (C) Binding kinetics between HCY-NBD and GSTM2 measured by Biacore T200. (D) GSTM2 enzymatic activity kinetics was measured in vitro following exposure to either DMSO solvent control or HCY-NBD.

Techniques:

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD binds to GSTM2 and stabilize its protein levels (A) Three-dimensional schematic diagram of the interaction between HCY-NBD and GSTM2. The yellow and gray structures represent the 3D conformation of GSTM2 protein, while HCY-NBD is depicted as a green stick model. Molecular docking simulations predict binding affinity between GSTM2 and HCY-NBD with a binding energy of −7.52 kcal/mol. (B) Two-dimensional interaction diagram between HCY-NBD and GSTM2. Green dashed lines indicate hydrogen bonds, light green dashed lines represent carbon-hydrogen bonds, and pink dashed lines denote hydrophobic interactions. (C) Binding kinetics between HCY-NBD and GSTM2 measured by Biacore T200. (D) GSTM2 enzymatic activity kinetics was measured in vitro following exposure to either DMSO solvent control or HCY-NBD. GS-DNB refers to the product of the GSTM2 enzymatic activity assay. (E) HUVEC cells were treated with increasing glucose concentrations (0, 10, 30, 60 mM) for 24 h, followed by quantification of GSTM2 mRNA levels via qPCR. (F) HUVECs were treated with HCY-NBD (0, 1, 3, 5 μM) for 24 h under HG (30 mM) conditions, the GSTM2 mRNA expression were analyzed by qPCR. (G – H) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the GSTM2 protein levels were determined by Western blot. (I – J) HUVECs were treated with HCY-NBD (0, 1, 3, 5 μM) for 24 h under HG (30 mM) conditions, the GSTM2 protein levels were determined by Western blot. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: Recombinant GSTM2 protein (HY–P75152A, MCE) was subjected to buffer exchange using PD MiniTrapTM G-25 (28-9180-07, GE Healthcare) to eliminate Tris-based primary amines.

Techniques: Binding Assay, Activity Assay, In Vitro, Solvent, Control, Enzyme Activity Assay, Expressing, Western Blot

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD up-regulates GSTM2 sulfenylation and inhibits GSTM2 ubiquitination (A-B) HUVECs were pretreated with 80 μM HDX for 1h, then the cells were treated with HCY-NBD (0 or 5 μM) and HDX (0 or 80 μM) under HG (30 mM) conditions for 24 h. Protein levels of GSTM2 were analyzed by Western blot. (C – D) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the sulfenylation levels of GSTM2 were measured by Western blot. (E – F) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the ubiquitination levels of GSTM2 were determined by Western blot. (G – H) HUVECs were transfected with GFP-GSTM2, and HA-Ub (Wild-type, K48-only, or K63-only), exposed to designated treatments, and assessed for GFP-GSTM2 ubiquitination levels via Western blot in all experimental groups. (I – L) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the glutathionylation levels of GSTM2 were determined by Western blot. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: Recombinant GSTM2 protein (HY–P75152A, MCE) was subjected to buffer exchange using PD MiniTrapTM G-25 (28-9180-07, GE Healthcare) to eliminate Tris-based primary amines.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: Cys174 is a critical residue for HCY-NBD-mediated regulation of GSTM2 sulfenylation and non-lysine ubiquitination (A-B) HUVECs were transfected with wide type (WT) and four distinct cysteine-to-alanine mutants (C87A, C115A, C174A, and C87-115-174A) of GSTM2, exposed to designated treatments, and assessed for GSTM2 sulfenylation levels via Western blot in all experimental groups. (C) HEK-293T cells were transfected with a GSTM2-overexpressing plasmid for 48 h. Ubiquitination at Cys174 was detected via LC-MS/MS, with MS/MS analysis demonstrating a characteristic +114.043 Da mass shift (C [114.043]). Score, 131.0. (D – E) HUVECs were transfected with wide type (WT) and 174 cysteine-to-alanine(C174A) mutants of GSTM2, exposed to designated treatments, and assessed for GSTM2 ubiquitination levels via Western blot in all experimental groups. (F – G) HUVECs were transfected with GFP-GSTM2-WT, GFP-GSTM2-C174A, and HA-Ub (WT, K48-only), exposed to designated treatments, and assessed for GFP-GSTM2 ubiquitination levels via Western blot in all experimental groups. (H–K) HUVECs were transfected with GFP-GSTM2-WT or GFP-GSTM2-C174A 24 h, then treated with cycloheximide (2.5 μM) and designated treatments for the indicated times, followed by Western blotting. (L – M) HUVECs were transfected with wide type (WT) and 174 cysteine-to-alanine(C174A) mutants of GSTM2, exposed to designated treatments, and assessed for HA levels via Western blot in all experimental groups. H–K: Two-way ANOVA was performed, others: one-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, n = 3.

Article Snippet: Recombinant GSTM2 protein (HY–P75152A, MCE) was subjected to buffer exchange using PD MiniTrapTM G-25 (28-9180-07, GE Healthcare) to eliminate Tris-based primary amines.

Techniques: Residue, Ubiquitin Proteomics, Transfection, Western Blot, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD inhibits high glucose-induced senescence of HUVECs (A-D) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, the protein levels of p21, p16 and p53 were determined by Western blot. (E – F) Cell cycle analysis by flow cytometry for HUVECs in different groups. (G – H) HUVECs were exposed to 0 or 5 μM HCY-NBD under either normal-glucose (5.5 mM) or HG (30 mM) conditions for 24 h, SA- β -gal staining was performed to observe senescence in HUVECs. (I – L) HUVECs were transfected with GSTM2 siRNA at a final concentration of 50 nM, while control groups were transfected with scramble siRNA at the same concentration. After 24 h, cells cultured under a HG (30 mM) model were treated with 0 or 5 μM HCY-NBD for 24 h. Protein levels of p21 were analyzed by Western blot and SA- β -gal staining was performed to observe senescence in HUVECs. (M – N) HUVECs were transfected with wide type (WT) and four distinct cysteine-to-alanine mutants (C87A, C115A, C174A, and C87-115-174A) of GSTM2, exposed to designated treatments, and assessed for protein levels of p21 via Western blot in all experimental groups. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3, n = 4(E–F).

Article Snippet: Recombinant GSTM2 protein (HY–P75152A, MCE) was subjected to buffer exchange using PD MiniTrapTM G-25 (28-9180-07, GE Healthcare) to eliminate Tris-based primary amines.

Techniques: Western Blot, Cell Cycle Assay, Flow Cytometry, Staining, Transfection, Concentration Assay, Control, Cell Culture

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: HCY-NBD inhibits vascular senescence in db/db mice (A) Experimental Workflow Diagram. (B) HCY-NBD was administered intraperitoneally to db/db mice (0, 1, or 5 mg/kg per injection, every other day) over an 8-week period. Body weights were recorded weekly, n = 5. (C–H) Following 8 weeks of treatment, mice were humanely euthanized. Major organs (heart, liver, spleen, lungs, and kidneys) were excised and weighed using an analytical balance. Bar charts depict organ-to-body weight ratios, calculated as (organ weight/total body weight) × 100 %, n = 5. (I) Histopathological evaluation of heart, liver, spleen, lung, and kidney tissues was performed on H&E-stained sections to characterize microstructural organization and injury phenotypes, n = 4. (J – L) Thoracic aortic segments were subjected to en face immunofluorescence staining to assess p21 levels within CD31-positive endothelial cells and CD31 levels. Nuclei were counterstained with DAPI. n = 4. (M – N) Vascular senescence in thoracic aortas was detected using SA- β -gal staining, n = 3. (O–P) Protein expression levels of GSTM2 in thoracic aortas of db/db mice were detected via immunohistochemistry, n = 5. (Q – R) Immunofluorescence analysis detected protein expression levels of GSTM2 colocalized with CD31 in thoracic aortas of db/db mice, n = 4. One-way ANOVA followed by Tukey's post-hoc test was performed, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Recombinant GSTM2 protein (HY–P75152A, MCE) was subjected to buffer exchange using PD MiniTrapTM G-25 (28-9180-07, GE Healthcare) to eliminate Tris-based primary amines.

Techniques: Injection, Mass Measurement, Staining, Immunofluorescence, Expressing, Immunohistochemistry

Journal: Redox Biology

Article Title: Small molecule HCY-NBD stabilizes GSTM2 via cys174 sulfenylation to attenuate high glucose induced endothelial cell senescence and calcification

doi: 10.1016/j.redox.2026.104058

Figure Lengend Snippet: Schematic presentation of the new approach to enhance endogenous GSTM2 level that inhibits high glucose-induced senescence and calcification in vascular endothelial cells.

Article Snippet: Recombinant GSTM2 protein (HY–P75152A, MCE) was subjected to buffer exchange using PD MiniTrapTM G-25 (28-9180-07, GE Healthcare) to eliminate Tris-based primary amines.

Techniques: