36a (bay-069)  (Eurofins)

Journal: Research

Article Title: Vascular Smooth Muscle Cell-Specific BCAT2 Deficiency Attenuates Diabetic Atherosclerotic Calcification via Histone Propionylation

doi: 10.34133/research.1052

Figure Lengend Snippet: BCAT2 inhibitor impedes diabetic atherosclerotic calcification. (A) Docking model of BAY-069 (BCAT2 inhibitor) and BCAT2. (B) qRT-PCR analysis of BCAT2 gene expression in the aorta of NC or DM ApoE −/− mice supplemented with DMSO or BAY-069 (0.3 mg/kg). n = 6. (C) Western blot image and analysis showing α-SMA, SM22α, BMP2, RUNX2, and BCAT2 protein expression level in the aorta of NC or DM ApoE −/− mice supplemented with DMSO or BAY-069 (0.3 mg/kg). n = 6. (D) BCAT2 enzyme activity in the aorta of indicated mice. n = 6. (E and F) Biochemical measurement of aortic calcium content and ALP activity for indicated mice. n = 6. (G) Representative images of alizarin red S staining in the whole aorta for indicated mice. (H) Micro-CT was used to detect aortic calcification for indicated mice. (I) Representative images and quantification of von Kossa staining in aortic arch and aortic root tissue sections, and immunohistochemistry staining of RUNX2 in aortic root tissue sections. Scale bars, 200 μm for von Kossa staining and 50 μm for immunohistochemistry staining. n = 6. (J) Western blot image and analysis showing α-SMA, SM22α, BMP2, RUNX2, and BCAT2 protein expression level in Movas supplemented with DMSO or BAY-069 under NM or OM treatment for 14 d. n = 6. (K) Representative images of alizarin red S staining. Scale bars, 200 μm. n = 6. (L) Quantification of calcium deposits. n = 6. Data are presented as mean ± SD. ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus DMSO.

Article Snippet: BAY-069 (catalog no. HY-148242) and C646 (catalog no. HY-13823) were obtained from MedChemExpress (New Jersey, USA).

Techniques: Quantitative RT-PCR, Gene Expression, Western Blot, Expressing, Activity Assay, Staining, Micro-CT, Immunohistochemistry

Journal: Investigative Ophthalmology & Visual Science

Article Title: BCAT1 Activation Reprograms Branched-Chain Amino Acid Metabolism and Epigenetically Promotes Inflammation in Diabetic Retinopathy

doi: 10.1167/iovs.66.6.59

Figure Lengend Snippet: Bcat1 is inhibited by Plk4-mediated phosphorylation. ( A ) Flag–BCAT1 was overexpressed in rMC-1 cells. Cells were incubated either in normal glucose (5.5-mM) with mannitol (24.5-mM) as an osmotic control or in high glucose (30-mM). Flag–BCAT1 was immunoprecipitated for the enzymatic activity assay. Immunopurified protein was further subjected to western blotting to determine serine/threonine phosphorylation (p-S/T), tyrosine phosphorylation (p-Y), lysine acetylation (ace-K), and lysine methylation (me-K). Bcat1 activity was normalized to immunopurified Flag protein. ( B ) Volcano plot showing differentially expressed protein kinases between WT and db/db retinas. ( C ) Flag–BCAT1 was co-expressed with HA-tagged BMPR1B, MASTL, or PLK4 in rMC-1 cells. Flag–BCAT1 was immunoprecipitated. Its enzymatic activity was analyzed and normalized to Flag protein. p-S/T of Bcat1 and p-Bcat1 T333 was determined. ( D ) rMC-1 cells overexpressing Flag–BCAT1 was cultured either in normal glucose (5.5-mM) with mannitol (24.5-mM) as an osmotic control or in high glucose (30-mM). Flag–BCAT1 was immunoprecipitated. Plk4 protein and Bcat1 T333 phosphorylation levels were determined by western blotting. ( E ) Plk4 and Bcat1 protein expression and p-Bcat1 T333 in the retinas of WT and db/db mice were determined by western blotting. ( F ) The correlation between Bcat enzymatic activity and p-Bcat1 T333 levels from mouse retinas is shown. Pearson correlation was determined. ( G ) WT and db/db mice were treated with or without BAY-069. Retina vessels were stained with Evans Blue to visualize vascular leakage. White arrows indicate the vascular leakage. Scale bar : 1 mm. ( H ) The working model shows that Bcat1 activity is increased in diabetic retinopathy, which is negatively regulated by Plk4. Increased Bcat1 activity promotes BCAA catabolism, leading to a reduction in α-KG levels. α-KG reduction results in higher H3K4me3 levels and overexpression of Il6 , Tnf-α , and Gfap , accelerating diabetic retinopathy. All data are shown as mean ± SD from at least three independent experiments. * P < 0.05, ** P < 0.01; n.s., no significant change.

Article Snippet: Then, 1 μL of Bcat inhibitor (1 mmol/L, BAY-069, #HY-148242; MedChemExpress, Monmouth Junction, NJ, USA), 1 μL of Bcat1-specific inhibitor (100 μmol/L or 200 μmol/L, ERG240, #HY-W193545A; MedChemExpress), or PBS (mock) was intravitreally injected under general anesthesia with ketamine (80 mg/kg) and xylazine (4 mg/kg).

Techniques: Phospho-proteomics, Incubation, Control, Immunoprecipitation, Enzyme Activity Assay, Western Blot, Methylation, Activity Assay, Cell Culture, Expressing, Staining, Over Expression