s shown. F: DNA pulldown assay. NRA1 was overexpressed in HEK293T cells, which had been treated with or with out AQ. Cell extracts were incubated together with the NBRE DNA within the HMGCR gene promoter and analyzed by immunoblot analysis with anti-NR4A1 antibody. Data in a are expressed because the mean SEM, and statistical analysis was conducted by Students t-test or ANOVA with Tukey’s honest considerable distinction post hoc test. P 0.005; P 0.0005 by Student’s t-test. #P 0.05; ##P 0.01 compared with handle (AQ = 0 M) by Tukey’s post hoc test. DAPI, 4,6-diamidino-2-phenylindole.conversion to TG by the action of GPAT, LPAAT, PAP, and DGAT (16, 26) (Fig. 4C). Thus, we also analyzed the impact of AQ on fatty acid synthesis and subsequent storage lipid conversion as a result of accumulated lipid vesicles. Although ACC1 expression was not changed by AQ treatment, FASN was prominently enhanced by AQ at the transcriptional level in each TM3 and major Leydig cells (Fig. 4D, E). Moreover, the lipidmodifying enzymes GPAT, LPAAT, and PAP weren’t impacted by AQ, whereas DGAT was considerably enhanced by AQ in Leydig cells (Fig. 4F). These benefits indicate that AQ considerably increased lipid biogenesis, specifically fatty acids and storage lipid TG, resulting in accumulation of lipid vesicles. AQ changes cellular lipid composition and enhances TG accumulation in Leydig cells Considering that AQ increases lipid accumulation in Leydig cells, we attempted to analyze cellular lipid composition employing a lipidomics approach. Principal component evaluation plot revealed that AQ distinctively changed the6 J. Lipid Res. (2021) 62cellular lipid composition of Leydig cells (Fig. 5A). Extensive modifications in lipid composition were observed in Leydig cells soon after remedy with AQ, as visualized by a heatmap (Fig. 5B). LC/MS-based lipid analysis confirmed that 67.three and 62.0 of total lipids were identified in vehicle- and AQ-treated Leydig cells, respectively, but AQ decreased structural lipids and enhanced storage lipids (Fig. 5C). Probably the most abundant structural lipids, PCs, were decreased in proportion in AQ-treated cells, whereas the percentage in the TG storage lipid was significantly elevated by AQ therapy. The ratio of Pc:PE was slightly but substantially enhanced in AQ-treated Leydig cells, reflecting adequate membrane integrity and cell viability (27). Further quantitative analysis showed that the general amount of total lipids was drastically elevated in Leydig cells soon after AQ treatment, showing precisely the same quantitative amount of structural lipids regardless of the lower proportion (Fig. 5D). Interestingly, the amount of COX-1 Inhibitor Formulation intracellular TG was significantly elevated in Leydig cells after treatment with AQ, which was also consistentFig. four. Increased lipid accumulation in AQ-treated Leydig cells. A: TM3 cells were treated with AQ and subjected to BODIPY staining. B: Quantitation of BODIPY staining intensity. C: The method for fatty acid synthesis and lipid biogenesis. D: TM3 cells have been incubated with AQ, and relative transcript amount of ACC1 was determined c-Rel Inhibitor Source following normalization with actin level. E: TM3 cells and primary Leydig cells were treated with AQ for 24 h, and relative transcript amount of FASN was determined by quantitative real-time PCR analysis. F: The relative transcript levels of lipogenic genes were determined in TM3 Leydig cells. Information in B, D, E, and F are expressed because the mean SEM. Statistical analysis was performed by ANOVA with Tukey’s honest significant differenc