ML792 disrupts SMAD4 SUMOylation-dependent nuclear translocation in TGFβ2-stimulated lens epithelial cells (LECs). (IMAGE)
Caption
(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, TGFβ2 (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.
Credit
Min Hou, Yujie Ding, Xuan Bao, Liangping Liu, Yulan Wang, Mingxing Wu
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