Fig 2: Increased cellular lipid levels are fueled by enhanced lipid uptake. a LNCaP cells were treated for the indicated times with Enz (10 µM) including the final 72 h in the presence of 13C-acetate. Incorporation of 13C into cholesterol via lipogenesis was measured by GC/MS (n = 3, mean ± SD). b Protein (blue) and mRNA (gray) expression of indicated enzymes was measured by quantitative proteomics with isobaric mass tag labels and microarray analysis, respectively (n = 3, mean ± SD). c LNCaP cells were treated as described in a, and cellular uptake of indicated fluorescent lipid probes was measured by qSCI based on the mean fluorescence intensity (MFI) of NBD-cholesterol, LDL(DiL), acetylated-LDL(DiL), or d NBD-PE and PC-A2 in LNCaP and C4-2B cells (n > 9000 cells, mean ± SD, representative of 2 independent experiments). e Fold-change mRNA expression changes of indicated lipid transporters relative to D0 of Enz-treatment were calculated from the microarray data described in Fig. 2a (top panel) or measured by qRT-PCR (bottom panel). f LNCaP cells were treated for the indicated times with Enz (10 µM), and protein expression of lipid transporters SCARB1 (left) and LDLR (right) was measured using immunofluorescence microscopy (IgG = antibody control, n > 9000 cells, mean ± SD, representative result from 2 independent experiments). g LNCaP cells were treated with Enz (10 µM) or grown in androgen-depleted media (CSS, charcoal-stripped serum) for the indicated times, and FA uptake was measured by qSCI based on the MFI of cellular Bodipy-C16:0 (n > 9000 cells, mean ± SD, representative result from 3 independent experiments). h LNCaP cells were treated for the indicated times with Enz (10 µM), and fatty acid content was analyzed by GC/MS FAME (n = 2, mean ± SD). Statistical analysis: a–h one-way ANOVA followed by Dunnett’s multiple comparisons test compared to D0; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 3: Effects of hesperetin on LXRa signal in THP-1 macrophages. THP-1 macrophages were treated with hesperetin (10 µmol/L, 50 µmol/L, 100 µmol/L) and/or T0901317 (10 µmol/L) for 24 h. Control cells were cultured for the same time without any treatments. Total proteins and RNAs were extracted and analyzed using western blot and real-time RT-PCR, respectively. (a and c) The effects of hesperetin on the levels of the LXRa protein and mRNA. (b and d) The best effective concentration of hesperetin of 100 µmol/L was used to further validate the hesperetin-induced activation of LXRa. (e–g) The effects of hesperetin on the levels of the ABCA1, ABCG1, and SR-BI proteins. The results are presented as the means ± SD from three independent experiments performed in triplicate. * p < 0.05 and ** p < 0.01 compared with control; ? p < 0.05 and ?? p < 0.01 compared with treatment with T0901317

Fig 4: Effects of metformin on SRA, CD36, AEBP1, SR-B1 and ABCG1 expressions in ox-LDL-treated macrophages. (A) Western blot analysis was used to measure the expression levels of SRA, CD36, AEBP1, SR-B1 and ABCG1 proteins following application of the indicated treatments; GAPDH was used as a control for the standardization of the total cellular protein. (B) Quantitative analysis of SRA levels. (C) Quantitative analysis of CD36 levels. (D) Quantitative analysis of AEBP1 levels. (E) Quantitative analysis of SR-B1 levels. (F) Quantitative analysis of ABCG1 levels. The data are expressed as mean ± standard deviation and are representative of three independent experiments. **P<0.01 vs. the PMA group, ***P<0.001 vs. the PMA group, #P<0.05 vs. the ox-LDL group, ##P<0.01 vs. the ox-LDL group and ###P<0.001 vs. the ox-LDL group. SRA, scavenger receptor A; CD36, cluster of differentiation 36; AEBP1, adipocyte enhancer-binding protein 1; SR-B1, scavenger receptor B1; ABCG1, ATP binding cassette transporter G1; ox-LDL, oxidized low-density lipoprotein; PMA, phorbol 12-myristate 13-acetate.

Fig 5: Vitamin D related proteins in several tissues at delivery in rats treated during pregnancy with 25-OH-D3 (experimental diet) or vitamin D3 (control diet). (A) Maternal liver: vitamin D receptor (VDR), fatty acid translocase (FAT/CD36) (P = 0.059), and Scavenger Receptor 1 (SR-B1); (B) placenta: VDR, FAT/CD36, and SR-B1; (C) fetal brain at delivery: VDR (P = 0.086) and glutamate decarboxylase (GAD). T-test significant differences *P < 0.05.