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. 2024 Nov 12;22:541. doi: 10.1186/s12964-024-01913-2

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 8 — Bromoxib exhibits selectivity towards proteins in the mitochondrial fatty acid β-oxidation pathway, leading to significant changes in the lipid composition of Ramos cells and affecting palmitate oxidation. Ramos cells were treated for (A) 30 min and (B) 24 h with DMSO (0.1% v/v; negative control), 10 µM or 40 µM bromoxib. The protein levels of the five stabilized proteins of the mitochondrial fatty acid β-oxidation protein cluster: acyl-CoA synthetase long chain family member 4 (ACSL4), hydroxyacyl-CoA dehydrogenase complex subunits A and B (HADHA (p90, p132) and HADHB), enoyl-CoA hydratase (ECH1) and acyl-CoA dehydrogenase very long chain (ACADVL) were determined via immunoblotting. GAPDH served as a loading control. (A) and (B) on the left side: representative immunoblots of three independent biological replicates are shown. On the right side: the respective quantification of the immunoblots as described in Materials and Methods. Error bars = mean ± SD of three independent experiments. Statistical analysis: 1-way ANOVA with Dunnett’s multiple comparison test; (**** = p ≤ 0.0001). In (C) and (D), Ramos cells were treated for 30 min with 10 µM bromoxib and DMSO (0.1% v/v; solvent control) and lipidomics was performed for the lysates (six biological replicates) on: (C) lysophosphatidylcholine (LPC), phosphatidylcholine (PC), phosphatidylethanolamine (PE), cardiolipin (CLN), phosphatidylserine (PS), phosphatidylinositol (PI) and free cholesterol (FC). The lysates were also analyzed for the different fatty acids (D) such as diacylglycerols (DGs), triacylglycerols (TGs), cholesteryl esters (CE) and free fatty acids (FFA). Error bars = mean ± SD of six independent experiments. In (E), the total and partial palmitate oxidation were analyzed in Ramos cells treated with DMSO (0.1% v/v), 10 µM bromoxib (five biological replicates) for 30 min. [¹⁴C]-CO₂ as well as [¹⁴C]-acid soluble metabolites were detected after metabolization as measures for the total and partial palmitate oxidation. Error bars = mean ± SD of five independent experiments. Statistical analysis: unpaired two-tailed t-test; (**** = p ≤ 0.0001)