ml792  (Cambridge Bioscience)

Journal: Genes & Diseases

Article Title: Blockage of SUMO E1 enzyme inhibits ocular lens fibrosis by mediating SMAD4 SUMOylation

doi: 10.1016/j.gendis.2025.101827

Figure Lengend Snippet: Pharmacological inhibition of SUMO E1 attenuates TGFβ 2 -driven epithelial–mesenchymal transition (EMT) and prevents anterior subcapsular cataract (ASC) progression. (A) FHL124 lens epithelial cells (LECs) were treated with 0.1% DMSO, 10 μM ML792, and 10 μM Ginkgolic acid (GA), along with or without the treatment of 10 ng/mL TGFβ 2 for 24 h. Immunoblot analysis of EMT markers, fibronectin, Collagen I, SLUG, and SNAIL proteins was performed. β-Tubulin served as the loading control. (B) Densitometric quantification of (A). One-way ANOVA with Bonferroni correction; ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (C–F) Global SUMOylation profiling in treatment groups from (A). (C) SUMO1 conjugate immunoblot. (E) SUMO2/3 conjugate immunoblot. (D, F) Quantification analysis of SUMOylation levels (normalized to GAPDH and β-tubulin). One-way ANOVA with Bonferroni correction; ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (G) Ex vivo rat lens organ culture model. Macroscopic lens opacity assessment after 7-day treatments: vehicle (0.1% DMSO), TGFβ 2 (10 ng/mL), ML792 (10 μM), and TGFβ 2 plus ML792. Bottom: histopathological analysis (hematoxylin-eosin staining) and fibrotic marker immunohistochemistry staining (fibronectin/α-SMA). Scar bar: 200 μm. (H, I) Immunoblot validation of fibrotic markers in lens epithelium from (G). GAPDH served as the loading control. One-way ANOVA with Bonferroni post-hoc test; ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (J) In vivo therapeutic efficacy in C57BL/6J mice: intracameral injection into the anterior ocular chamber with vehicle (0.1% DMSO diluted in PBS) and ML792 (10 μM diluted in PBS) administered immediately post-capsular injury ( n = 6 biological replicates/group). 7-day endpoints: slit-lamp imaging (red arrow indicated plaques; scale bar: 0.5 mm) and immunohistochemistry staining of α-SMA protein (scale bar: 200 μm). (K, L) Immunoblot analysis of SUMOylation status in murine lens epithelium from the therapeutic intervention groups described in (J). (M) Densitometric quantification of SUMO conjugation levels (SUMO1, SUMO2/3). Data were normalized to β-tubulin and GAPDH. Unpaired Student's t -test; ∗ P < 0.05 and ∗∗∗ P < 0.01. (N, O) Immunoblot analysis of fibrotic markers (fibronectin/α-SMA) in murine lens epithelium from (J), followed by densitometric quantification. GAPDH served as the loading control. Unpaired Student's t -test; ∗ P < 0.05 and ∗∗∗ P < 0.001.

Article Snippet: Drug preparation was as follows: TGFβ 2 (#8406LC, CST, USA) in PBS containing 0.1% bovine serum albumin; ginkgolic acid (#22910, TargetMol, China) and ML792 (#S8697, Selleck, China) in dimethyl sulfoxide (DMSO, #196055, MP Biomedicals, USA).

Techniques: Inhibition, Western Blot, Control, Ex Vivo, Organ Culture, Staining, Marker, Immunohistochemistry, Biomarker Discovery, In Vivo, Drug discovery, Injection, Imaging, Conjugation Assay

Journal: Genes & Diseases

Article Title: Blockage of SUMO E1 enzyme inhibits ocular lens fibrosis by mediating SMAD4 SUMOylation

doi: 10.1016/j.gendis.2025.101827

Figure Lengend Snippet: ML792 disrupts SMAD4 SUMOylation-dependent nuclear translocation in TGFβ 2 -stimulated lens epithelial cells (LECs). (A – F) FHL124 LECs were treated with or without TGFβ2 (10 ng/mL, 2 h). Triple immunofluorescence staining of SMAD4 (green), SUMO1 (red)/SUMO2/3 (red), and DAPI (nuclei, blue) shows spatiotemporal dynamics of SMAD4-SUMO colocalization. (A, D) SMAD4-SUMO1/SUMO2/3 immunofluorescence staining and colocalization scatterplot. (B, E) Pearson's r analysis of colocalization performed by Image J. n = 9 replicates per group. (C, F) Quantification of nuclear SMAD4 intensity. n = 30 cells in (C) and n = 44 cells in (F). Unpaired Student's t -test; ∗ P < 0.05 and ∗∗∗ P < 0.001. (G, H) Flag-SMAD4 immunoprecipitation in engineered FHL124 LECs overexpressing Flag-SMAD4. Treatments were 0.1% DMSO, TGFβ 2 (10 ng/mL), ML792 (10 μM), or their combination for 2 h. (G, H) Whole-cell lysates were blotted with anti-Flag and anti-SMAD4 (INPUT). Cell lysates were immunoprecipitated with anti-Flag, followed by SUMO1 immunoblotting (G) and SUMO2/3 immunoblotting (H). (I, J) Subcellular fractionation analysis. (I) Immunoblots of cytoplasmic/nuclear SMAD4 after 8 h treatments in FHL12.4 LECs. (J) Quantification was normalized to GAPDH (cytoplasm) and lamin A/C (nucleus). One-way ANOVA with Bonferroni correction; ns, not significant; ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (K, L) SMAD4 nuclear translocation analysis. (K) Triple immunofluorescence staining SMAD4 (red), F-actin (Phalloidin, green), and DAPI (nuclei, blue) in LECs treated as indicated in (I). Scar bar: 20 μm. (L) Nuclear SMAD4 fluorescence intensity quantification. n = 30 cells per group. One-way ANOVA with Bonferroni post-hoc test; ∗ P < 0.05 and ∗∗∗ P < 0.001.

Article Snippet: Drug preparation was as follows: TGFβ 2 (#8406LC, CST, USA) in PBS containing 0.1% bovine serum albumin; ginkgolic acid (#22910, TargetMol, China) and ML792 (#S8697, Selleck, China) in dimethyl sulfoxide (DMSO, #196055, MP Biomedicals, USA).

Techniques: Translocation Assay, Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Fractionation, Fluorescence

Journal: Genes & Diseases

Article Title: Blockage of SUMO E1 enzyme inhibits ocular lens fibrosis by mediating SMAD4 SUMOylation

doi: 10.1016/j.gendis.2025.101827

Figure Lengend Snippet: Mechanistic schema of SUMO E1-mediated SMAD4 SUMOylation in lens fibrogenesis. In TGFβ/SMAD signaling, the SAE1/UBA2 heterodimer (SUMO E1) catalyzes SMAD4 SUMOylation at Lys113/159 residues, enhancing nucleocytoplasmic trafficking efficiency of the SMAD complex. SUMOylated SMAD4 accumulates in the nucleus, stabilizing the transcriptional machinery of epithelial–mesenchymal transition (EMT)-related genes ( e.g. , SNAIL , SLUG , FN1 , COL1A1 ), thereby amplifying fibrotic gene expression. Sustained SUMOylation drives lens epithelial cell (LEC) transdifferentiation, characterized by α-SMA expression and extracellular matrix overproduction, culminating in anterior subcapsular cataract (ASC) progression. Pharmacological inhibition of SUMO E1 by ML792 blocks SMAD4 SUMOylation, disrupting nuclear translocation and abrogating pro-fibrotic transcriptional programs.

Article Snippet: Drug preparation was as follows: TGFβ 2 (#8406LC, CST, USA) in PBS containing 0.1% bovine serum albumin; ginkgolic acid (#22910, TargetMol, China) and ML792 (#S8697, Selleck, China) in dimethyl sulfoxide (DMSO, #196055, MP Biomedicals, USA).

Techniques: Gene Expression, Expressing, Inhibition, Translocation Assay

Journal: Endocrinology

Article Title: Glucocorticoid Receptor and SUMO Fluctuations in Response to Pulsatile Glucocorticoids In Vitro and in Male Rat Brains

doi: 10.1210/endocr/bqaf140

Figure Lengend Snippet: Ligand-induced GR downregulation requires GR SUMOylation. (A) HCT treatment (20/50/100nM for 6 hours) of GFP-GR-wt-transfected A549 cells decreased GFP-GR protein levels (n = 3-4); one-way ANOVA with Dunnett's multiple comparisons test results shown. (B) HCT treatment (20/50/100nM for 6 hours) of GFP-GRmutSUMO-transfected A549 cells did not decrease GFP-GRmutSUMO protein levels (n = 4; one-way ANOVA, Dunnett's multiple comparisons test results not statistically significant. (C) Western blot of A549 cells transfected with GFP-GR-wt and treated with vehicle or HCT (50nM for 6 hours); lane 3 and 4 cells were pretreated with ML792 (1µM) or vehicle (0.1% (v/v) DMSO) for 18 hours prior to HCT addition. (D) Representative Western blot showing the effect of treatment ± HCT (50nM for 6 hours) on cycloheximide-pretreated A549 cells: untransfected cells, cells transfected with GFP-GR-wt and cells transfected with GRmutSUMO. (E) Densitometry results showing GFP-GR-wt downregulation by HCT (50nM for 6 hours) in cycloheximide-pretreated cells (50 µg/mL 1 hour prior to HCT addition) was blocked by GR SUMO site mutations. Data represent GAPDH-normalized Western blot densitometry measurements of transfected GFP-GR from HCT-treated relative to vehicle-treated cells (n = 3/4); unpaired t-test results shown. (F) Cycloheximide chase assay showing GFP fluorescence assay data from A549 cells transfected GFP-GR-wt/GFP-GRmutSUMO and treated with HCT (50nM) for up to 4 hours (n = 3/4) relative to expression at time = 0. (G) The 3 hours timepoint of the cycloheximide chase assay showing GFP-GR downregulation due to HCT. A549 cells were transfected with GFP-GR constructs encoding wild-type GR, the full triple mutant (GRmutSUMO), or partial mutants with SUMO site mutations only in the N-terminal (GRmutS12) or only in the C-terminal (GRmutS3) (n = 4). Western densitometry data were normalized to GAPDH, in graphs A, B, and E. Graphs are presented as mean ± SEM. Effect of treatment * P < .05, ** P < .01.

Article Snippet: Hydrocortisone, dexamethasone, and cycloheximide were from Sigma (Gillingham, UK); ML792 (HY-108702-5 mg) from Cambridge Bioscience (Cambridge, UK).

Techniques: Transfection, Western Blot, Fluorescence, Expressing, Construct, Mutagenesis

Journal: Endocrinology

Article Title: Glucocorticoid Receptor and SUMO Fluctuations in Response to Pulsatile Glucocorticoids In Vitro and in Male Rat Brains

doi: 10.1210/endocr/bqaf140

Figure Lengend Snippet: Glucocorticoid withdrawal increases endogenous GR protein expression. (A) Timecourse showing downregulation of GR-HiBiT following exposure to 50nM HCT, in GR-HiBiT A549 cells. Relative fluorescence units (RFU) represent Nano-Glo® HiBiT lytic detection assay luminescence readings after background subtraction and normalization to luminescence at t = 0 minutes. Data represent mean ± SEM, n = 3/4. (B) Timecourse showing downregulation of GR-HiBiT following pretreatment with 1µM ML792/vehicle (DMSO 0.1% [v/v]) for 1 hour and exposure to 50nM HCT, in GR-HiBiT A549. Data represent mean ± SEM, n ≥ 3. (C) Effect of ML792 (ratio ML792-treated/vehicle-treated) on GR-HiBiT expression after 1 hour HCT treatment, in GR-HiBiT A549 cells. Cells were pretreated with vehicle (DMSO 0.1% [v/v])/1 µM ML792, ± cycloheximide, for 1 hour then exposed to 50nM HCT for 1 hour. Data represent mean ± SEM, n ≥ 9, 2-way ANOVA (2 treatments) with Tukey's multiple comparisons test. (D) GR-HiBiT expression following withdrawal of hormone, in GR-HiBiT A549 cells pretreated with 50nM HCT for 18 hours. Cells were subjected to ×3 media exchanges to wash out (−) or maintain (+) HCT, then further incubation for 2/4/6 hours in the absence/presence of HCT. Data represent mean ± SEM, n = 3, 2-way ANOVA (treatment and time) with Tukey's multiple comparisons test. (E) Timecourse showing effect of ML792 on upregulation of GR-HiBiT following hormone withdrawal, in GR-HiBiT A549 cells pretreated with 50nM HCT for 18 hours. Pretreatment with 1µM ML792/vehicle (DMSO 0.1% [v/v]) for 1 hour was followed by media replacements to wash out HCT. Data represent mean ± SEM n = 3. Effect of treatment * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: Hydrocortisone, dexamethasone, and cycloheximide were from Sigma (Gillingham, UK); ML792 (HY-108702-5 mg) from Cambridge Bioscience (Cambridge, UK).

Techniques: Expressing, Fluorescence, Detection Assay, Incubation