Detection of transfected (exogenous) and endogenous APE1/Ref-1 forms.

<p>(A) Western Blot detection of transfected flagged APE1/Ref-1 proteins using anti-Flag antibody (left panel) and detection of endogenous (black arrow) and flagged APE1/Ref-1 (white arrow) using anti-APE1/Ref-1 antibody (right panel). (B) Microphotography of flagged APE1/Ref-1 cellular localization in the two cell lines obtained after transfection. In blue: Hoescht stained nuclei. In red: mitotracker-red stained mitochondria. In green: FITC detected flagged APE1/Ref-1 forms. Merge is obtained overlapping all three images; orange/yellow indicates a mitochondrial localization of the flagged proteins.</p

APE1/Ref-1 mRNA relative levels determined by Real Time RT-PCR in HCC affected patients.

<p>(A) APE1/Ref-1 mRNA level increase in HCC with respect to DLC for each patient. (B) APEX1 mRNA fold change in HCC with respect to DLC. (* p = 0.002 vs. DLC). (C) SLC/DLC ratio and HCC/DLC ratio for each patient. Abbreviations: DLC: distal liver cirrhosis; SLC: surrounding liver cirrhosis; HCC: hepatocellular carcinoma; NL: normal liver.</p

APE1/Ref-1 mRNA and protein levels in Hepatoma cell lines with respect to IHH.

<p>(A) Real Time RT-PCR analysis expressed as fold change in mRNA expression of APE1/Ref-1 in hepatoma cell lines as compared to normal hepatocytes (IHH). Data are expressed as mean± SD of five different experiments from 5 different batchs of cells *p = 0.0035 vs. Huh7. (B) WB analysis for APE1/Ref-1 in total extracts of hepatoma cell lines and in normal hepatocytes. Left: representative WB for APE1/Ref-1 detection. Right: band density quantification graph. Samples were normalized to α -tubulin and protein levels in Huh-7 and JHH-6 are relative to IHH cells, data are expressed as mean±SD of five different experiments from 5 different batchs of cells. (* p = 0.001 vs. Huh7).</p

Intracellular localization of APE1/Ref-1 in hepatoma cell lines.

<p>(A) Immunofluorescence for APE1/Ref-1 in IHH, Huh-7 and JHH6. In red: propidium iodide (PI) stained nuclei. In green: FITC-immunodetected APE1/Ref-1. APE1/Ref-1 is localized both in the nucleus and in the cytoplasm of each cell line. Merge is obtained overlapping the two images; orange/yellow indicates nuclear localization of APE1/Ref-1 while green indicates cytoplasmic localization of this protein. (B) Western blot analysis for APE1/Ref-1 in IHH, Huh-7 and JHH6 nuclear and cytoplasmic fractions. Representative WB for APE1/Ref-1 in IHH, Huh-7 and JHH6 nuclear and cytoplasmic fractions. (C) Band density quantification graph for nuclear (grey bars) and cytoplasmic fractions (black bars). Samples were normalized to α-tubulin and α-p84 respectively and protein levels in Huh-7 and JHH-6 are relative to IHH cells, data are expressed as mean ± SD of three different experiments from 5 different batches of cells.</p

APE1/Ref-1 exerts hepatocytes protection against oxidative insult and apoptosis.

<p>(A) Hydrogen peroxide cytotoxicity on transfected IHH measured by MTT assay (black diamond: IHH/p3X; black triangle: IHH/pRef-1). Data are expressed as mean ± SD of three different experiments. (* = different from IHH/p3X; p<0.05). (B) FACS analysis of apoptotic rate after UV irradiation. Cells were treated with UV exposure and after 6 hours FACS analysis for AnnexinV staining was performed to assess the percentage of cells undergoing apoptosis. Left Panel: example of FACS analysis graphs for apoptotic rate at 6 hours after UV irradiation (FL1-H: AnnexinV-FITC; FL2-H: propidium iodide). Right Panel: The bar plot shows the AnnexinV-FITC positive cells mean ± SD of three different experiments from three different batches of cells (black bars: IHH/p3X; grey bars: IHH/pRef-1). (* = different from IHH/p3X). (C) Immunocytochemical detection of Bax activation and Cytochrome C release and positive cell counting. Figures represent an example of immunocytochemical detection of Bax activation (upper figure) and Cytochrome C release (lower figure) after UV treatment. In blue: Hoescht stained nuclei. In red: mitotracker-red stained mitochondria. In green: FITC detected Cytocrome C. Merge is obtained overlapping all three images, orange/yellow indicates a mitochondrial localization of BAX and Cytocrome C. After BAX activation Cytochrome C is released into the cytoplasm losing its mitochondrial localization. Bar graphs represent the percentage of cells with BAX activation and Cytocrome C release after UV irradiation (grey bars: untreated; black bars: 6 hours of UV exposure). Data are reported as mean ± SD of three different experiments. (*p<0.01 vs. IHH/p3X). UT: untreated.</p

Real Time PCR and Sirius red-Fast Green staining.

<p><b>(a)</b> mRNA expression of Col1A1 and <b>(b)</b> α-SMA. <b>(c)</b> Collagen protein quantification.</p

Histopathological findings.

<p>Histological analysis of the liver at each experimental checkpoint times according to Brunt's classification. Data is expressed as score/grade and between brackets the occurrence.</p

Hepatic and aortae histological findings.

<p>Hepatic histopathological analysis. <b>(b-e)</b> Representative images of Hematoxylin-Eosin staining progression of HFHC group over the time <i>vs</i>. CTRL <b>(a).</b> Higher magnification of the dashed area <b>(f-j). (l-o)</b> Gömöri trichrome staining for collagen of HFHC group over the time <i>vs</i>. CTRL <b>(k)</b>. <b>(p, q)</b> Representative picture of Hematoxylin & Eosin staining in aortae of controls <b>(p)</b> and HFHC group <b>(q).</b> In all pictures scale bar is 100 μm.</p

Glucose and insulin quantification in males and females.

<p>^a p<0.05, ^b p<0.01, ^c p<0.001, ns: not statistically relevant. Mean ± SD <i>vs</i>. age and sex matched CTRL.</p

Lipid profile analysis and serum ALT.

<p><b>(a)</b> Total cholesterol. <b>(b)</b> HDL-C. <b>(c)</b> LDL-C. <b>(d)</b> Triglycerides. <b>(e)</b> ALT. Data are expressed as mean ± SD. Statistical significance was calculated <i>vs</i>. age and sex matched CTRL. * p<0.05, ** p<0.01, *** p<0.001.</p