Fig 1: Inhibition of MGAT2, but not MGAT3, reduced DG and TG synthesis and TG secretion. (A) HepG2 cells were pre-incubated for 1 h with or without the MGAT2, MGAT3 or both inhibitors. Cells were then incubated with 5 µCi [3H]glycerol and 0.25 mM OA. After 5 h, cells were harvested and the incorporation of radioactivity into TG, DG and phospholipids was determined. (B) HepG2 cells were incubated with the MGAT2 (25 µM) or MGAT3 (50 µM) inhibitors for 5 h. Cells were harvested and DGAT activity in crude mitochondrial fractions was determined. Data are the mean ± standard deviation of two experiments performed in duplicate. (C) HepG2 cells were incubated with DMEM in the presence or absence of the MGAT2 or MGAT3 inhibitors (separately and together). After 1 h, cells were incubated with 5 µCi [3H]glycerol and 0.25 mM oleic acid for 4 h. Cells were then washed and incubated an additional 4 h in DMEM. The amount of [3H]TG secreted into the media was determined. Data are the mean ± standard deviation of two experiments performed in duplicate. Values for DMSO-treated controls: DG (1.9 × 103 dpm/mg cell protein), TG (11.2 × 103 dpm/mg cell protein), PL (30.2 × 103 dpm/mg cell protein), secreted TG (0.89 × 103 dpm/mg cell protein). (D) Effect of MGAT inhibition on apoB secretion. ApoB in the culture media was quantified by ELISA after incubation of HepG2 cells with 0.25 mM OA for 6 h in the presence of the MGAT inhibitors, as described in A. Data are the mean ± standard deviation of three independent experiments. *p < 0.01; **p < 0.001, ***p < 0.05.
Fig 2: Confocal images using red fluorescent silica nanoparticles.a–g Use fluorescent Si-NP and h–n uses fluorescent Si-NP' both added directly after sample incubation. All the images of membranes had anti-ApoAI as the capture antibody except for g and n that had Anti-ApoB. The images show the state of membrane for sample containing a 1 pM, b 10 pM, c 100 pM, d 1 nM and e 10 nM of PON1-HDL and h 1 pM, i 10 pM, j 100pM, k 1 nM and l 10 nM of HDL-P. A protein cocktail containing rePON1, reApoAI and HSA (1:40:1000) that contained an equivalent protein in 10 nM PON1-HDL and total HDL respectively, i.e., f 10 nM rePON1 + 400 nM reApoAI + 10000 nM HSA and m 1 nM rePON1 + 40 nM reApoAI + 1000 nM HSA were used as controls. g and n used 10 nM of HDL as sample but with anti-ApoB, thus providing no signal. Scale bars are 100\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu$$\end{document}µm. Imaging was done once for each case.
Fig 3: Blind study with clinical samples comparing PON1-HDL to other biomarkers from different platforms.a PON1-HDL using NGEMS (AUC = 0.99). b PON1-HDL using ELISA-1 (AUC = 0.90). c Total PON1 using sandwich ELISA (AUC = 0.83). d PON1 activity assay using rate of reaction with substrate (AUC = 0.67). e Non-HDL cholesterol (AUC = 0.645), f HDL cholesterol (AUC = 0.64) and g cholesterol ratio (AUC = 0.65) from cholesterol assay. h Triglyceride levels from triglyceride assay (AUC = 0.65). Total HDL particle concentration HDL-P from i competitive ELISA (AUC = 0.62), j ELISA-2 (AUC = 0.775), k NGEMS as sum of PON1-HDL and PON1-free HDL (AUC = 0.645) and l 1H-NMR (AUC = 0.61). m PON1-free HDL from NGEMS (AUC = 0.52). n ApoAI from sandwich ELISA (AUC = 0.69). o ApoB from sandwich ELISA (AUC = 0.60). p OxLDL from commercial ELISA (AUC = 0.67). p-values are calculated from unpaired parametric one-tailed t-test with Welch’s correction. AUC values calculated from ROC plots (see Supplementary Fig. 3 for ROC plots). Each datapoint was average of several replicates. Every sample in the figures are measured in triplicates except in e–g where they are duplicated. Each plot is a standard box and whisker plot with central line being the median, box being the 25th and 75th quantile and whiskers representing 0th and 100th quantile. Both controls and CAD groups had ten samples (n = 10) each.
Supplier Page from Thermo Fisher Scientific for Human ApoB ELISA Kit