Additional file 2: Figure S2. of Oleate ameliorates palmitate-induced reduction of NAMPT activity and NAD levels in primary human hepatocytes and hepatocarcinoma cells

Palmitate decreases cell viability after 24 h. After stimulation of HepG2 cells with palmitate and oleate for 4 h and 24 h at the indicated concentrations, cell viability was measured via WST-1 assay. Data were referred to control (serum free medium) and represent three independent experiments performed in triplicates shown as means ± SEM. ***p < 0.05 compared to control. (JPEG 51 kb

Additional file 1: Figure S1. of Oleate ameliorates palmitate-induced reduction of NAMPT activity and NAD levels in primary human hepatocytes and hepatocarcinoma cells

Solvent controls do not influence cell viability as well as intracellular NAMPT and NAD levels. HepG2 cells were stimulated with serum-free medium with and without DMSO (dilution factor: 1:1′000‘000) and serum free medium with 1% free fatty acid -BSA with and without NaOH (dilution 1:200). Neither DMSO nor NaOH altered cell viability measured by WST-1 assay (A), intracellular NAD levels measured by HPLC (B), NAMPT mRNA expression or protein abundance measured by qPCR or Western blot analysis (C,D), respectively, enzyme activity (E) and lipid accumulation stained with Oil-red O (F). Data were normalised to control (serum free medium) which was set 1. Data represent two or three independent experiments shown as means ± SEM. FFA: free fatty acid. (JPEG 691 kb

Sequences of Primer and Probes used for <i>real-time</i> PCR (TaqMan).

<p><i>NAMPT (</i>nicotinamide phosphoribosyltransferase, also known as PBEF, visfatin); <i>p21</i>; housekeeping genes <i>beta-ACTIN</i>, <i>TBP</i> (TATA-box-binding protein) and <i>HPRT</i> (hypoxanthine phophoribosyltransferase).</p

SIRT1 overexpression in HepG2 cells reversed resveratrol-induced SIRT1 inhibition, NAMPT release and S-phase arrest.

<p>A) SIRT1 was transiently overexpressed in HepG2 cells [2.0 µg plasmid/0.5x10⁶ cells] using the expression vector pECE_Flag-SIRT1 from addgene (plasmid 1791; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091045#pone.0091045-Brunet1" target="_blank">[35]</a>). Lysates of cells transfected with the empty vector pECE (mock-control) (1) or pECE Flag-SIRT1 vector (2) were used for Western Blot analysis. B) mock-transfected (mock-control) and Flag-SIRT1 transfected HepG2 cells were stimulated with resveratrol [50, 100 µM Resv.] for 24 h and Western Blot analysis of acetylated p53 (K382), p21 and GAPDH was performed. Densitometric anaylsis of acetylated p53 of three independent Western Blots is shown. Data were normalised to non-transfected HepG2 cells stimulated with resveratrol alone which was set 1. C) To analyse the effect of SIRT1 overexpression on resveratrol-induced NAMPT release, supernatant of mock-transfected and Flag-SIRT1 transfected HepG2 cells stimulated with or without resveratrol [100 µM] were used to measure eNAMPT level. One representative Western blot out of 3 independent experiments is shown. D) Cell viability of mock-transfected and Flag-SIRT1 transfected HepG2 cells treated with resveratrol [100 µM] (black bars) was measured using WST-1 assay (n = 3). Data were normalised to untreated mock-control which was set 1. E) mock-transfected (white bars) and Flag-SIRT1 transfected HepG2 cells (black bars) were stimulated with resveratrol [25, 50 µM] for 24 h. Percentage of cells in the S-phase were measured by PI staining and FACS analysis. All data are shown as mean± SEM (n = 4). The difference between two groups was evaluated using unpaired Student’s <i>t</i>-test (##p<0.01 mock-transfected cells compared to mock-transfected cells treated with resveratrol (white bars, mock-control), **p<0.01, ***p<0.001 Flag-SIRT1 transfected cells treated with resveratrol (black bars) compared to resveratrol-treated mock-transfected cells (white bars).</p

Resveratrol differentially regulates p53 acetylation and SIRT1 protein level in HepG2 cells and primary human hepatocytes.

<p>Acetylation of p53 (K382) in A) HepG2 cells (n = 4) and B) primary human hepatocytes (n = 3) was evaluated by Western Blot. Densitometric analysis of at least three independent experiments is shown. Data are represented as mean± SEM and statistical analysis was performed using one-way ANOVA and the Bonferroni post hoc test (*p<0.05, n.s. not significant). As a downstream target of acetylated and activated p53, the expression of p21 was analysed by Western Blot. As positive control for SIRT1 inhibition, EX527+TSA was used. SIRT1 protein expression was analysed by Western Blot in C) HepG2 cells and D) primary hepatocytes and densitometric analysis was performed. GAPDH was used as loading control, respectively. One representative blot out of at least 3 independent experiments is shown.</p

Resveratrol activates apoptotic mechanisms in hepatocarcinoma cells.

<p>Cells were treated with resveratrol or serum-free medium (con) for 24 h. Activation of p53 through phosphorylation at serine residue 15 and cleavage of caspase-3 in A) HepG2 cells, B) Hep3B cells and C) primary human hepatocytes were analysed by Western Blot. GAPDH was used as loading control. One representative blot out of at least 3 independent experiments is shown.</p

SIRT1 inhibition downregulates NAMPT activity and induces NAMPT release in HepG2 cells.

<p>HepG2 cells were treated with EX527+TSA [20 µM EX527+1 µM TSA] or serum-free medium (con) for 24 h. Measurement of A) NAMPT enzymatic activity (n = 3). Counts (cpm) were normalised to µg total protein in each sample (*p<0.05). B) NAD level were determined by HPLC (n = 5) and normalised to total protein amount in each sample. C) Supernatant of EX527 treated HepG2 cells was used for determination of eNAMPT level. One representative Western blot out of 3 independent experiments is shown.</p

Effects of resveratrol on NAMPT release and NAMPT mRNA expression.

<p>Cells were stimulated with resveratrol in serum-free medium for 24 h. Supernatants of resveratrol treated A) HepG2 cells (n = 7) and B) primary human hepatocytes (n = 3) were used for quantifying extracellular NAMPT protein amount using a specific eNAMPT ELISA. eNAMPT protein concentration was normalised to the total protein amount. <i>NAMPT</i> mRNA expression in resveratrol treated C) HepG2 cells (n = 5) and D) primary human hepatocytes (n = 4) was quantified by qRT-PCR and normalised to housekeeping genes. <i>NAMPT</i> gene expression was then related to its expression in serum-free control medium (0), which was set 1. Data are represented as mean± SEM and statistical analysis was performed using one-way ANOVA and the Bonferroni post hoc test (*p<0.05; ***p<0.001; n.s. not significant). E) Supernatant of resveratrol [100 µM] or serum-free medium (con) treated HepG2 cells was used to measure NAMPT enzymatic activity and extracellular NAMPT protein levels. Counts (cpm) measured by NAMPT enzyme assay were referred to densitometric data of NAMPT protein levels in the supernatant of the same sample. Data were then normalised to serum-free control medium which was set 1. Data are shown as mean± SEM. The difference between these two groups was evaluated using unpaired Student’s <i>t</i>-test (***p<0.001).</p

Resveratrol reduces cell proliferation and induces cell cycle arrest and apoptosis in hepatocarcinoma cells which is absent in primary human hepatocytes.

<p>Cell viability of A) primary human hepatocytes (n = 2), HepG2 and Hep3B cells (n = 3) after stimulation with resveratrol for 24 h. Data were normalised to serum-free medium control which was set 1. B) Cell cycle distribution of HepG2 cells treated with resveratrol for 24 h. A representative result is shown out of three independent experiments. A representative dot plot is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091045#pone.0091045.s002" target="_blank">Fig. S2B</a>. C) Annexin V/PI apoptosis assay of HepG2 (n = 3) and Hep3B cells (n = 3) treated with resveratrol for 24 h. D) A representative dot plot of the Annexin/PI staining in HepG2 cells is shown including the mean percentage of An+ and double An+/PI+ cells of three independent experiments. Data are shown as mean± SEM and statistical analysis was performed using one-way ANOVA and the Bonferroni post hoc test (*p<0.05; **p<0.01 compared to serum-free medium).</p

NAMPT and SIRT1 expression in hepatocarcinoma cells and primary human hepatocytes.

<p>A) mRNA expression and B) protein expression of NAMPT and SIRT1 in primary human hepatocytes (n = 7), HepG2 cells (n = 8) and Hep3B cells (n = 3). Representative Western Blot is shown out of three independent experiments. Measurement of C) intracellular NAD levels (left panel, primary hepatocytes n = 4, HepG2 cells n = 6), basal NAMPT enzymatic activity (middle panel, primary hepatocytes n = 3, HepG2 cells n = 4) and extracellular NAMPT (eNAMPT) levels (right panel, primary hepatocytes n = 3, HepG2 cells n = 6) in primary human hepatocytes and HepG2 cells. Data are shown as mean± SEM. Difference between two groups was evaluated using unpaired Student’s <i>t</i>-test (*p<0.05, **p<0.01, ***p<0.001).</p