Fig. 3
PPARα depletion alters lipid metabolism in ACE-overexpressing macrophages. A–G TPMs were incubated with vehicle (ethanol) or 200 μM OA for 16 h. A, B In vitro lipid uptake assay. The intracellular lipid content was determined via Lipi-Deep Red (LDR) staining followed by flow cytometry analysis. Representative histograms (A) and MFI values (fold change) (B) of LDR signals in TPMs. C, D CD36 expression. Representative histograms (C) and MFI values (fold change) (D) of CD36 expression in TPMs measured by flow cytometry. E–G Lipid consumption. TPMs were treated with 200 μM OA at 37 °C for 16 h and then washed and cultured in media (without OA) for 6, 12, or 18 h. Intracellular lipids were stained with LDR and quantified via flow cytometry at each time point. E Time-dependent lipid reduction. F Fluorescence microscopy images of intracellular lipids in TPM immediately following OA loading (0 h) or after 18 h without OA. G Lipid reduction rate at 18 h post-OA exposure. H Gene expression profile of OA-treated TMPs. The TPM samples were cultured with 200 μM OA for 16 h, after which the isolated total RNA was subjected to real-time PCR. Gene expression was quantified via the ∆Ct method. I, J Lipid peroxidation. TPMs were incubated with 200 μM OA for 16 h, and lipid peroxidation was subsequently measured by staining with a peroxidation probe and flow cytometry. Representative histogram (I) and MFI values (fold change) (J) of lipid peroxidation. K, L ROS production. TPMs were incubated with ethanol or 200 μM OA for 16 h. Cytosolic ROS and mitochondrial ROS (mtROS) were measured by flow cytometry with H2DFCDA and MitoSOXTM, respectively. Representative histograms (K) and MFI values (fold change) (L) of cytosolic ROS levels in TPMs. Representative histograms (M) and MFI values (fold change) (N) of mtROS levels. O Measurement of the intracellular ATP concentration. TPMs were incubated with ethanol or OA as described above, and then, intracellular ATP was quantified via the luminescence-based assay CellTiter-Glo 2.0. The ATP concentrations were calculated via the standard curve method. P–R Real-time metabolic analysis of TPM by Seahorse. P Transition of the oxygen consumption rate (OCR) in the TPMs during analysis. Q, R Basal respiration and maximal respiration of TPMs. The cumulative data are shown as the means ± SEMs of five to six samples from two independent experiments. All MFI values are represented as fold changes (the average value of WT was used for base = 1). One-way ANOVA was used to analyze the significance of the data. *p < 0.05, **p < 0.01 and ***p < 0.01. ns indicates not significant
