Fig 1: YY1 binds with VEGFA and Snail1, and ubiquitination and degradation of YY1 are mediated by Smurf2.A VEGFA and Snail1 levels were detected by western blotting in ARPE-19 cells transfected with shNC or shYY1. B The binding of YY1 on the promoter of VEGFA and Snail1 was analyzed by ChIP in ARPE-19 cells transfected with shNC or shYY1. C Luciferase activity was measured in ARPE-19 cells co-transfected with empty vector (EV) or YY1 overexpression vector and WT-Snail1, Mut-Snail1, WT-VEGFA, or Mut-VEGFA. D Smurf2 and YY1 levels were detected by western blotting after Co-IP of YY1 antibody. E YY1 and Smurf2 levels were measured via western blotting in ARPE-19 cells transfected with siSmurf2 or Scramble. F Western blotting analysis of YY1 levels in WCL derived from HEK293T cells with or without MG132 treatment. G YY1 and Smurf2 levels were examined by western blotting in ARPE-19 cells transfected with siSmurf2 or Scramble before treatment of cycloheximide (CHX) for different time points. H Smurf2-overexpressed HEK293T cells were treated with CHX for 0, 15, 30, 60, 120, and 240 min in the presence of MG132 (a proteasome inhibitor), followed by the detection of YY1 using western blotting. I Expression vectors encoding Flag-YY1 and HA-ubiquitin were co-transfected into HEK293T cells transfecting Myc-Smurf2 and cell lysates were subjected for Co-IP using anti-Flag, which was followed by western blotting using anti-HA antibody. For each analysis, three technical replicates were performed and three biological independently performed replicates are included, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: MG infection induces the secretion of HMGB1 in HD-11 cells. (A) The protein expression of HMGB1 was detected by WB. Tubulin, YY1, ß-actin, or Na+/K+ ATPase are considered as the loading control. In addition, GAPDH was shown as the negative control of membrane protein. (B) The mRNA expression of HMGB1 HD-11 cells. GAPDH was used for normalization (n = 4). (C) Left: HMGB1 levels in the supernatant were measured by HMGB1 Elisa kit (n = 4). Right: The expression of HMGB1 was detected in the exosomes derived from MG-infected cells (n = 3). (D) Confocal images of HMGB1 (red) and DAPI (bule) in HD-11 cells. White arrows: membrane-localized HMGB1 (scale bar, 2 µm). * p < 0.05 and ** p < 0.01 show statistically significant difference compared to the 0 h group. Black dots in bar graph represent datasets from independent experiments.
Fig 3: The inhibitive effect of YY1 knockdown on expression of VEGFA and Snail1, and HG-induced EMT and cell permeability of ARPE-19 cells.ARPE-19 cells were transfected with shNC or shYY1 before treatment of HG. A YY1 level was detected by western blotting in the treated cells. B, C The migratory ability was examined via wound-healing analysis and transwell assay in the treated cells. D Occludin, E-cadherin, and Vimentin abundances were detected via immunofluorescence in the treated cells. E Cell permeability was analyzed by FITC-dextran assay in the treated cells. F VEGFA, Snail1, Occludin, E-cadherin, N-cadherin, and Vimentin abundances were measured by western blotting in the treated cells. For each analysis, three technical replicates were performed and three biological independently performed replicates are included, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: MiR-195 knockdown upregulates Smurf2 to inhibit YY1 and represses HG-induced EMT and cell permeability of ARPE-19 cells.ARPE-19 cells were transfected with inhibitor NC or miR-195 inhibitor, and were then stimulated or not stimulated by HG. A MiR-195 abundances were measured via qRT-PCR in the transfected cells. B Smurf2 and YY1 abundances were measured via western blotting in the transfected cells. C, D Cell migration was measured using wound-healing analysis and transwell assay in the treated cells. E Occludin, E-cadherin, and Vimentin abundances were determined by immunofluorescence in the treated cells. F Cell permeability was evaluated via FITC-dextran assay in the treated cells. G VEGFA, Snail1, Occludin, E-cadherin, N-cadherin, and Vimentin levels were detected via western blotting in the treated cells. For each analysis, three technical replicates were performed and three biologically independently performed replicates are included, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5: The expression of miR-195, YY1, VEGFA, Snail1, and Smurf2 in STZ-induced diabetic mice and HG-stimulated ARPE-19 cells.A The pathology of retinas from control or model group was analyzed by HE staining. B MiR-195, Smurf2, YY1, VEGFA, Snail1, Occludin, E-cadherin, N-cadherin, and Vimentin levels were detected via qRT-PCR or western blotting in the retinas from control or model group. C Cell morphology was observed under microscope in ARPE-19 cells after stimulation of different doses of glucose. Magnification: ×100. D VEGFA, Snail1, Occludin, E-cadherin, and Vimentin levels were examined by immunofluorescence in ARPE-19 cells in control or HG group. Magnification: ×200. E MiR-195, Smurf2, YY1, VEGFA, Snail1, Occludin, E-cadherin, N-cadherin, and Vimentin levels were examined by qRT-PCR or western blotting in ARPE-19 cells after 0, 24, 48, and 72 h of HG exposure. For each analysis, three technical replicates were performed and three biological independently performed replicates are included, *p < 0.05, **p < 0.01, ***p < 0.001.
Supplier Page from Abcam for Anti-YY1 antibody