Journal: Molecular cell

Article Title: A Metabolic Basis for Endothelial-to-Mesenchymal Transition

doi: 10.1016/j.molcel.2018.01.010

Figure Lengend Snippet: (A) Representative Western blot and quantification of phosphorylated SMAD2 (pSMAD2) in the presence or absence of the TGFβ signaling inhibitor SB431542 following control or CPT1A knockdown (n=3 independent experiments). (B) Levels of acetyl-CoA in control or cytokine-treated endothelial cells (n=3 independent experiments). (C) Cytosolic acetyl-CoA levels can be increased by ACSS2-mediated acetate conversion or reduced by the ACLY chemical inhibitor SB-204990. ACLY, ATP citrate lyase; ACSS2, acyl-coenzyme A synthetase short-chain family member 2. (D) Acetate treatment inhibits cytokine-induced pSMAD2 (n=3 independent experiments). (E) Acetate treatment inhibits EndoMT (technical triplicates per condition, data are representative of one of three independent experiments). (F) Acetate treatment inhibits cytokine-induced EndoMT protein expression. (G) SB-204990 induces EndoMT (technical triplicates per condition, data represents one of three independent experiments). (H and I) Western blot analysis of wildtype SMAD7 protein levels with or without acetate or SB204990 (H) or expression of the K64/70A acetylation-defective SMAD7 mutant under these same conditions (I) (n=3 independent experiments). Data represents mean ± SEM, with significance determined by one-way ANOVA with Bonferroni’s multiple comparison test (A, D, E, H, I). * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: For chemical induction of EndoMT, subconfluent endothelial cells on fibronectin-coated 6-well plates were incubated with the ACLY inhibitor, SB-204990 (Tocris) at final concentration of 60 µM or with a mitochondrial citrate transport protein (CTP) inhibitor (Sigma-Aldrich) at a final concentration of 1 mM.

Techniques: Western Blot, Expressing, Mutagenesis