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: Cells were lysed in 0.5% NP-40 buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 10% glycerol) containing protease inhibitors (#P2714, Sigma–Aldrich, Missouri, USA) on ice for 5 min. Lysate (2 mg) was precleared with control IgG (#2729, #53484, CST) at 4 °C for 2 h. Immunoprecipitation was performed at 4 °C overnight using anti-Flag antibody/Nano-Agarose beads (#FNM-25-500, NuoyiBio, Tianjin, China), anti-SUMO1 or anti-HA antibody with protein A/G Magnetic beads (#HY-K0202, MedChemExpress, New Jersey, 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: SUMOylation site mutagenesis abolishes SMAD4-mediated epithelial–mesenchymal transition (EMT) in TGFβ 2 -stimulated lens epithelial cells (LECs). (A) Sanger sequencing validation of SMAD4 mutants. WT, wild-type; K113R, Lys113→Arg; K159R, Lys159→Arg. The black frames indicate WT and mutated codons. (B, C) SUMOylation capacity analysis in SMAD4 mutants. (B) FHL124 LECs stably overexpressed empty vector and flag-SMAD4 variants treated with TGFβ 2 (10 ng/mL, 2 h). Whole-cell lysates were immunoblotted with anti-Flag and anti-SMAD4. β-Tubulin served as the loading control. The cell lysates were immunoprecipitated with anti-Flag nano beads, followed by immunoblotting for SUMO1, SUMO2/3, and Flag antibody. (C) Quantification of SMAD4 expression (Input lysates). One-way ANOVA with Bonferroni correction; ns, not significant; ∗∗∗ P < 0.001. (D, E) SMAD4 nuclear translocation analysis. (D) Triple fluorescence imaging of Flag (SMAD4, red), F-actin (phalloidin, green), and DAPI (nuclei, blue) in engineered LECs treated with TGFβ 2 (10 ng/mL, 2 h). (E) Nuclear SMAD4 intensity quantification ( n = 15–18 cells/group). One-way ANOVA with Bonferroni post-hoc test; ∗∗∗ P < 0.001. (F, G) Functional consequence of double site mutant (K113 plus 159R) SMAD4 protein. (F) EMT marker immunoblotting 24 h after TGFβ 2 treatment in human LECs overexpressing empty vector, WT Flag-tagged SMAD4, or double site mutant Flag-tagged SMAD4. (G) Densitometric analysis from (F). β-Tubulin served as the loading control. One-way ANOVA followed by Bonferroni correction; ns, not significant; ∗ P < 0.05. ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Cells were lysed in 0.5% NP-40 buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 10% glycerol) containing protease inhibitors (#P2714, Sigma–Aldrich, Missouri, USA) on ice for 5 min. Lysate (2 mg) was precleared with control IgG (#2729, #53484, CST) at 4 °C for 2 h. Immunoprecipitation was performed at 4 °C overnight using anti-Flag antibody/Nano-Agarose beads (#FNM-25-500, NuoyiBio, Tianjin, China), anti-SUMO1 or anti-HA antibody with protein A/G Magnetic beads (#HY-K0202, MedChemExpress, New Jersey, USA).

Techniques: Mutagenesis, Sequencing, Biomarker Discovery, Stable Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Expressing, Translocation Assay, Fluorescence, Imaging, Functional Assay, Marker