Journal: iScience

Article Title: BRD3 PROTAC degrader targets H3K18ac to alleviate retinal microglia-driven uveitis

doi: 10.1016/j.isci.2025.114526

Figure Lengend Snippet: D072 exerts anti-inflammatory effects by specifically degrading BRD3 (A) The expressions of BRD2, BRD3, and BRD4 in control group, EAU group, and D072 treatment group of the retina. (B) The in vitro expression of BRD2, BRD3, and BRD4 under the intervention of the ubiquitin pathway. (C) Molecular docking of BRD3 and BRD4. (D) After knockdown of BRD3 in BV2 cells, the protein expression levels of INOS, COX2, and Galectin-3 receptor in response to LPS stimulation were measured. (A) Upper: representative western blot images of retinal BRD2, BRD3, and BRD4 in Control group, EAU+Vehicle group, and EAU+D072 group, respectively. Lower: quantification of the relative fold changes of the left ( n = 3/group; mean ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA). (B) Left: representative western blot images of BRD2, BRD3, and BRD4 in each group in BV2. Right: quantification of the relative changes of the left ( n = 3/group; mean ± SD; ∗∗∗p < 0.001; one-way ANOVA). (C) The binding modes of BRD3 and BRD4 with D072. (D) Western blotting analysis of the protein levels of iNOS, COX-2, Galectin-3, and BRD3 in different groups. Left: representative western blot images of iNOS, COX-2, Galectin-3, and BRD3 in different groups. Right: quantification of the relative fold changes of the left ( n = 3/group; mean ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA).

Article Snippet: BRD3 , Santa Cruz , sc-81202; RRID:AB_1119692.

Techniques: Control, In Vitro, Expressing, Ubiquitin Proteomics, Knockdown, Western Blot, Binding Assay

Journal: iScience

Article Title: BRD3 PROTAC degrader targets H3K18ac to alleviate retinal microglia-driven uveitis

doi: 10.1016/j.isci.2025.114526

Figure Lengend Snippet: The degradation of BRD3 changed the modification of histone acetylation (A) The CO-1P experiment of BRD3 and histone 3. (B) Changes in various histone modifications after D072 treatment. (C) The alteration of acetylation modification under drug treatment. (D) The changes in H3K18ac expression of retinal microglia under drug treatment. (A) Representative western blot images of immunoprecipitation IgG and BRD3 and IB: Histone3. (B) Left: representative western blot images of pan-antibody of different modifications, including acetylation, lactylation, succinylation, crotonylation, malonylation, and Histone 3. Right: quantification of the relative changes of the left ( n = 3/group; mean ± SD; ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA). (C) Left: representative western blot images of different site-specific acetylated antibodies. Right: quantification of the relative fold changes of the left ( n = 3/group; mean ± SD; ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA). (D) Representative immunofluorescent staining images of microglia (IBA-1, green) and H3K18ac (red) in the retinas of control, EAU+Vehicle, and EAU+D072 mice (white arrows represent double-positive cells). (Scale bars, 100 μm).

Article Snippet: BRD3 , Santa Cruz , sc-81202; RRID:AB_1119692.

Techniques: Modification, Expressing, Western Blot, Immunoprecipitation, Staining, Control

Journal: iScience

Article Title: BRD3 PROTAC degrader targets H3K18ac to alleviate retinal microglia-driven uveitis

doi: 10.1016/j.isci.2025.114526

Figure Lengend Snippet: The regulation of H3K18ac by BRD3 degradation is mediated by HDACs, and CCL5 is its target (A) The expression of several deacetylase enzymes (HDAC1, HDAC2, Sirt1, and Sirt3) in control group, LPS group, and D072 treatment group experiments in vitro . (B) The expression levels of SIRT1 under various treatments (LPS stimulation, D072 treatment, or siBRD3 knockdown), Sirt1 changes in proteins and mRNA. (C) The mRNA levels of cxcl10, ccl5, HK3, and nos2 in control group, LPS group, D072 treatment group, D072 + SIRT1 inhibitor (EX-527 treated) group in vitro . (D) The expression levels of CCL5, CXCL10, HK3, and NO2 under the intervention of each group of HDAC inhibitors. (Inhibitor terminology and function: SB: sodium butanoate [HDACs inhibitor], EX-527 [SIRT1 inhibitor], SIRT-IN-2 [SIRT1, 2, 3 inhibitors], SIRT6-IN-5 [SIRT6 inhibitor], and 97491 [SIRT7 inhibitor]). (E) The expression changes of H3K18ac under the intervention of each group of HDAC inhibitors. (Inhibitor terminology and function: SB: sodium butanoate [HDACs inhibitor], EX-527 [SIRT1 inhibitor], SIRT-IN-2 [SIRT1, 2, 3 inhibitors], SIRT6-IN-5 [SIRT6 inhibitor], and 97491 [SIRT7 inhibitor]). (A) Upper: representative western blot images of HDAC1, HDAC2, SIRT1, and SIRT3 in each group. Lower: quantification of the relative changes of the left ( n = 3/group; mean ± SD; ns, p > 0.05, ∗p < 0.05, ∗∗∗p < 0.001; one-way ANOVA). (B) Left: representative western blot images and the corresponding quantification of the relative protein levels of SIRT1 in each group. Right: quantification of the relative mRNA changes of the SIRT1 in different groups. ( n = 3/group; mean ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA). (C) Quantification of the mRNA level of Cxcl10 , Ccl5 , Hk3 , and inos after SIRT1 inhibitor EX-527 treatment. ( n = 3/group; mean ± SD; ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA). (D) Quantification of the mRNA level of Cxcl10 , Ccl5 , Hk3 , and inos under different HDAC inhibitor treatment. ( n = 3/group; mean ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA). Inhibitor terminology and function: SB: sodium butanoate (HDACs inhibitor), EX-527 (SIRT1 inhibitor), SIRT-IN-2 (SIRT1, 2, 3 inhibitors), SIRT6-IN-5 (SIRT6 inhibitor), and 97491 (SIRT7 inhibitor). (E) Quantification of the relative H3K18ac protein level under each treatment. ( n = 3/group; mean ± SD; ∗∗∗p < 0.001; one-way ANOVA). (F) Left: transwell images of different groups; scale bars: 100 μm. Right: quantification of migration cells under each treatment. ( n = 3/group; mean ± SD; ∗∗∗p < 0.001; one-way ANOVA). (G) Quantification statistics concentration of CCL5 in each group. ( n = 3/group; mean ± SD; ∗∗∗p < 0.001; one-way ANOVA).

Article Snippet: BRD3 , Santa Cruz , sc-81202; RRID:AB_1119692.

Techniques: Expressing, Histone Deacetylase Assay, Control, In Vitro, Knockdown, Western Blot, Migration, Concentration Assay