Journal: iScience

Article Title: Acetyl-CoA derived from hepatic mitochondrial fatty acid β-oxidation aggravates inflammation by enhancing p65 acetylation

doi: 10.1016/j.isci.2021.103244

Figure Lengend Snippet: Activation of ACLY supported increases in ac-p65 levels and inflammatory gene expression under lipid surplus (A) The levels of total and phosphorylated ACLY were examined by Western blot analysis and quantitated after treatment with different diets (n = 3). (B) The levels of total and phosphorylated ACLY were examined by Western blot analysis and quantitated after treatment with PA in fish hepatocytes (n = 3). (C) Inhibition of ACLY with SB-204990 decreased the content of acetyl-CoA in fish hepatocytes (n = 3). (D) Inhibiting ACLY with SB-204990 blocked the increase in ac-p65 induced by PA in fish hepatocytes (n = 3). (E) Inhibiting ACLY with SB-204990 attenuated the increase in inflammatory gene ( Tnf-α and Il-6 ) expression induced by PA in fish hepatocytes (n = 3). (F) Inhibiting Akt with MK2206 attenuated the increase in inflammatory gene ( Tnf-α and Il-6 ) expression induced by PA in fish hepatocytes (n = 3). (G) Inhibiting Akt with MK2206 attenuated PA-induced increase in p-ACLY in fish hepatocytes (n = 3). The results are presented as the mean ± SD and were analyzed using independent t-tests (∗p< 0.05).

Article Snippet: SB 204990 , MedChemExpress , CAT#HY-16450.

Techniques: Activation Assay, Expressing, Western Blot, Inhibition

Journal: iScience

Article Title: Acetyl-CoA derived from hepatic mitochondrial fatty acid β-oxidation aggravates inflammation by enhancing p65 acetylation

doi: 10.1016/j.isci.2021.103244

Figure Lengend Snippet: PA enhanced p65 acetylation to intensify inflammation in AML12 hepatocytes (A) The mRNA expression levels of inflammatory genes ( Cox-2 , Il-6 and Il-18 ) were analyzed after treatment with different concentrations of PA for 24 h (n = 3). (B) The acetyl-CoA levels were analyzed after 500 μM PA treatment for 24 h (n = 3). (C) The acetylation level of p65 was examined by Western blot analysis and quantitated after treatment with different concentrations of PA for 24 h (n = 3). (D) The mRNA expression levels of genes associated with fatty acid oxidation ( Cpt1α , Acaa2 and Ech1 ) were analyzed after treatment with different concentrations of PA for 24 h (n = 3). (E) Inhibition of fatty acid oxidation with etomoxir blocked the increase in ac-p65 induced by PA (n = 3). (F) Inhibition of fatty acid oxidation with etomoxir relieved the increase in Il-18 mRNA expression induced by PA (n = 3). (G) The levels of total and phosphorylated ACLY were examined by Western blot analysis and quantitated after treatment with different concentrations of PA for 12 h and 24 h (n = 3). (H) Inhibition of ACLY with SB-204990 blocked the increase in ac-p65 induced by PA (n = 3). (I) Inhibition of ACLY with SB-204990 attenuated the increase in Il-6 mRNA expression induced by PA (n = 3). (J) The mRNA expression levels of KATs ( Cbp , Gcn5 and Pcaf ) and Sirt1 were analyzed after 500 μM PA treatment for 24 h (n = 3). (K) Inhibition of GCN5 with MB-3 relieved the increase in Il-18 mRNA expression induced by PA (n = 3). (L) KDAC inhibitor treatment further increased the mRNA expression levels of inflammatory genes ( Il-6 and Il-18 ) under PA treatment (n = 3). The results are presented as the mean ± SD and were analyzed using independent t-tests (∗p< 0.05) and Tukey’s tests (bars bearing different letters are significantly different among treatments (p< 0.05)).

Article Snippet: SB 204990 , MedChemExpress , CAT#HY-16450.

Techniques: Expressing, Western Blot, Inhibition

Journal: iScience

Article Title: Acetyl-CoA derived from hepatic mitochondrial fatty acid β-oxidation aggravates inflammation by enhancing p65 acetylation

doi: 10.1016/j.isci.2021.103244

Figure Lengend Snippet:

Article Snippet: SB 204990 , MedChemExpress , CAT#HY-16450.

Techniques: Recombinant, Transfection, Activity Assay, Colorimetric Assay, Reporter Assay, Mutagenesis, Plasmid Preparation, Software

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

Journal: bioRxiv

Article Title: Targeted induction of de novo Fatty acid synthesis enhances MDV replication in a COX-2/PGE 2α dependent mechanism through EP2 and EP4 receptors engagement

doi: 10.1101/323840

Figure Lengend Snippet: A pathway interference approach to dissect the role of lipids in MDV replication. (A) Schematic FA synthesis pathway highlighting the relevant pharmacological inhibitors (red) and the respective enzymes (yellow box) as well as metabolites (green box) studied within FA synthesis pathway. (B) Fold change gene expression in CEFs mock-infected or MDV-infected are shown at 24, 48 and 72 hpi. Analysis of MDV viral titer (PFU/ml) in infected CEFs in the presence of (C) SB 204990 (1.28, 2.56 and 3.85 μM), (D) TOFA (0.03, 0.077, 0.154, 0.31, 0.77 and 1.54 μM) and (E) C75 (0.393, 1.97, 3.93 and 5.9 μM). Box and whisker plots demonstrating relative fold change in mRNA of (F) ACC and (G) FASN in MDV-infected CEFs cells treated with vehicle (Cont.) or TOFA+C75 (T/C) at 72 hpi. Analysis of MDV viral titer (PFU/ml) in infected CEFs in the presence of (H) malonyl-CoA with SB-204990, (I) malonyl-CoA with TOFA or (J) Palmitic acid (5, 12.5, 20 and 25 μM) (K) Palmitic acid with C75 (5.9 μM). (L) MDV (RB1B) genome copy number per 10 4 cells (MEQ gene with reference ovotransferrin gene) in the presence of SB 204990 (3.85 μM), TOFA (1.54 μM) or C75 (5.9 μM). *** (p = 0.0002) **** (p < 0.0001) indicates a statistically significant difference compared to control. NS indicates no significant difference. $ symbol indicates vehicle treated cells. All viral titer experiments were performed in 6 replicates and data is representative of 3 independent experiments.

Article Snippet: Chemicals: SB 204990 (Thermo Fisher Scientific, Paisley, UK), TOFA, C75, Clofibrate, Palmitic acid, SC-236 (Sigma-Aldrich, Dorset, UK), PGD 2 , PGI 2 , PGE 2α (Cambridge Bioscience, Cambridge, UK), SC-51322, TG-4-155 and ER-819762 (Bio-Techne Ltd., Abingdon, UK) were all reconstituted in DMSO.

Techniques: Expressing, Infection, Whisker Assay

Journal: bioRxiv

Article Title: Targeted induction of de novo Fatty acid synthesis enhances MDV replication in a COX-2/PGE 2α dependent mechanism through EP2 and EP4 receptors engagement

doi: 10.1101/323840

Figure Lengend Snippet: (A) Visualisation of cytoplasmic lipids in neutral lipid droplet organelles of CEFS (i) infected with MDV and (ii) mock-infected. Cell monolayer was fixed and stained with an Oil Red O staining kit and counterstained with hematoxylin and visualized using light microscopy (magnification 10X). Black arrows indicate lipid droplets. (B) Confocal microscopy imaging with maximum projection of Z-stacks for each channel demonstrating nuclear and cytoplasmic distribution of pRB1B UL35-GFP virus (green) and lipid droplets (red). Mock and pRB1B UL35-GFP infected cells were fixed at 72 hpi and stained with DAPI (nuclear stain) and the neutral lipid stain LIPIDTOX-568nm. Images 5 and 10 are 3-D representative images analysed using the IMARIS software. Z-stacks were analysed using the IMARIS spot function analysis tool to quantify the relative amount of lipid droplets per cell in infected CEFs and non-infected CEFs (50-90 cells). (C) The numbers of lipid droplets per cell in MDV-infected and mock-infected CEFs. The numbers of lipid droplets per cell in (D) mock infected (E) MDV-infected CEFs treated with SB 204990 (3.85 μM), TOFA (1.54 μM), C75 (5.9 μM), etomoxir (4.42 μM) and palmitic acid (25 μM) are shown. **** (p < 0.0001) indicates a statistically significant difference compared to the control. All experiments were performed in duplicates and data is representative of 3 independent experiments.

Article Snippet: Chemicals: SB 204990 (Thermo Fisher Scientific, Paisley, UK), TOFA, C75, Clofibrate, Palmitic acid, SC-236 (Sigma-Aldrich, Dorset, UK), PGD 2 , PGI 2 , PGE 2α (Cambridge Bioscience, Cambridge, UK), SC-51322, TG-4-155 and ER-819762 (Bio-Techne Ltd., Abingdon, UK) were all reconstituted in DMSO.

Techniques: Infection, Staining, Light Microscopy, Confocal Microscopy, Imaging, Software