Cell-based assay for the detection of chemically induced cellular stress by immortalized untransformed transgenic hepatocytes-1

<p><b>Copyright information:</b></p><p>Taken from "Cell-based assay for the detection of chemically induced cellular stress by immortalized untransformed transgenic hepatocytes"</p><p>BMC Biotechnology 2004;4():5-5.</p><p>Published online 19 Mar 2004</p><p>PMCID:PMC406386.</p><p>Copyright © 2004 Sacco et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p>ations of NaAsO(As1: 10M, As2: 5 × 10M, As3: 10M) and CdCl(Cd1: 10M, Cd2: 5 × 10M, Cd3: 10M). . Mean ± SE of viability of PHGH and MMH-GH5 at the indicated treatment conditions as determined by the Trypan blue dye exclusion method. These results were obtained from 6 independent experiments with treatments carried out in triplicate

Graphical representation of the proposed TGFbeta-induced mechanism of hepatocyte binucleation.

<p>Graphical representation of the proposed TGFbeta-induced mechanism of hepatocyte binucleation.</p

The <i>in vivo</i> inhibition of TGFbeta receptor hampers hepatocyte binucleation.

<p>(A) Experimental schedule for <i>in vivo</i> TGFbetaRI/II inhibitor (LY2109761) administration. (B) H&E of liver tissue from LY2109761-treated and control animals. In the lower panels are shown micrographs where binucleated and mononucleated cells are marked in red and green, respectively. A magnification of cells from the upper right panel was shown. (C) Percentage of binucleated cells of LY2109761-treated and control livers. Data are expressed as average values of counts of at least ten slices for each of four mice ± s.e.m. (number of counted cells: >1000 for each mouse). (***p value < 0.001). (D) Percentage of Ki-67 positive hepatocytes, analyzed by immunofluorescence on liver slices from LY2109761-treated and control mice. Data are expressed as average values of counts of at least five slices for each of four mice ± s.e.m. (number of counted cells: >400 for each mouse). (n.s. = not significant p value).</p

The TGFbeta1-induced cytokinesis failure is associated with the delocalization of active RhoA from the midbody.

<p>(A) Representative immunofluorescence of cytokinesis events with RhoA-GTP properly localized at the midbody (upper panel) or delocalized (middle and lower panels), considered for the count in both untreated hepatocytes and in TGFbeta-treated ones. In particular, the images refer to TGFbeta-treated hepatocytes. (B) Percentage of mitotic cells negative for RhoA-GTP signal at the midbody. Data are expressed as average values of three different experiments ± s.e.m (number of counted cells: >100 for each experimental condition) (*p value < 0.05). (C) Representative immunofluorescence of the cytokinesis events analysed in three independent experiments, showing the proper localization of Aurora B at the midbody upon TGFbeta1 treatment.</p

Ferritin Heavy Chain Is the Host Factor Responsible for HCV-Induced Inhibition of apoB-100 Production and Is Required for Efficient Viral Infection

Hepatic fat export occurs by apolipoprotein B-100-containing lipoprotein production, whereas impaired production leads to liver steatosis. Hepatitis C virus (HCV) infection is associated to dysregulation of apoB-100 secretion and steatosis; however, the molecular mechanism by which HCV affects the apoB-100 secretion is not understood. Here, combining quantitative proteomics and computational biology, we propose ferritin heavy chain (Fth) as being the cellular determinant of apoB-100 production inhibition. By means of molecular analyses, we found that HCV nonstructural proteins and NS5A appear to be sufficient for inducing Fth up-regulation. Fth in turn was found to inhibit apoB-100 secretion leading to increased intracellular degradation via proteasome. Notably, intracellular Fth down-regulation by siRNA restores apoB-100 secretion. The inverse correlation between ferritin and plasma apoB-100 concentrations was also found in JFH-1 HCV cell culture systems (HCVcc) and HCV-infected patients. Finally, Fth expression was found to be required for robust HCV infection. These observations provide a further molecular explanation for the onset of liver steatosis and allow for hypothesizing on new therapeutic and antiviral strategies

Tetraploid hepatocytes proliferate maintaining the poliploidy state in their progeny after TGFbeta1 withdrawal.

<p>(A) Immunofluorescence for E-Cadherin and Snail of TGFbeta1-treated, TGFbeta1-released and control MMH/E14 hepatocytes. Arrows indicate binucleated cells. (B) Transcriptional analysis by qRT-PCR of E-Cadherin and Snail in the indicated experimental conditions. Data are expressed as average values of three different experiments ± s.e.m. (ns = not significant p value), and plotted as ratio between treated and untreated (CTRL = 1) cells. (C) Percentage of binucleated cells in the indicated experimental conditions. Data are expressed as average values of three different experiments ± s.e.m. (*p value < 0,05). (D) Flow cytometry analysis for DNA content of cells cultured in the indicated experimental conditions.</p

Ferritin Heavy Chain Is the Host Factor Responsible for HCV-Induced Inhibition of apoB-100 Production and Is Required for Efficient Viral Infection

Hepatic fat export occurs by apolipoprotein B-100-containing lipoprotein production, whereas impaired production leads to liver steatosis. Hepatitis C virus (HCV) infection is associated to dysregulation of apoB-100 secretion and steatosis; however, the molecular mechanism by which HCV affects the apoB-100 secretion is not understood. Here, combining quantitative proteomics and computational biology, we propose ferritin heavy chain (Fth) as being the cellular determinant of apoB-100 production inhibition. By means of molecular analyses, we found that HCV nonstructural proteins and NS5A appear to be sufficient for inducing Fth up-regulation. Fth in turn was found to inhibit apoB-100 secretion leading to increased intracellular degradation via proteasome. Notably, intracellular Fth down-regulation by siRNA restores apoB-100 secretion. The inverse correlation between ferritin and plasma apoB-100 concentrations was also found in JFH-1 HCV cell culture systems (HCVcc) and HCV-infected patients. Finally, Fth expression was found to be required for robust HCV infection. These observations provide a further molecular explanation for the onset of liver steatosis and allow for hypothesizing on new therapeutic and antiviral strategies

Additional file 1: of CD90+ liver cancer cells modulate endothelial cell phenotype through the release of exosomes containing H19 lncRNA

(a) Characterization of isolated exosomes. Left panel: DSL for exosomes released by SKHep Middle panel: Western blot forTsg101 and HSC70 in SkHep cells and their relative exosomes. Right panel: Confocal microscopy analysis on HUVECs treated for 1, 3 and 6 hours with 5 mg/ml of SKHep-derived exosomes. HUVECs were stained with phalloidin Alexa Fluor (green), nuclear counterstaining was performed using DAPI (blue), exosomes were labelled with PKH26 (red). (b) Target analysis. Real time-PCR analysis on HUVECs treated for 18 h with 5 mg/ml of SkHep-derived exosomes. Normalized for b-actin the DDct were indicated as fold of induction respect to control (untreated cells). *p<0.05. (c) Tubulogenesis of HUVECs after exosomes treatment. Matrigel assay performed on HUVECs cells after 18 hour of 5 mg/ml SkHep-derived exosomes.  Left panel: phase contrast, magnification 20x. Right panel: quantification of matrigel assay expresses as length of cable as arbitrary unit **p<0.01. (d) Adhesion assay of SkHep cells on HUVECs. Left panel: phase contrast, magnification 10X. Right Panel: quantification of Huh7 or SKHep cells adherent on HUVECs, performed by counting the number of CD90+adherent cells (violet) per field ***p<0.001. (e) Analysis on H19 expression in exosomes. Real time-PCR analysis for H19 expression performed on SkHep-derived exosomes respect to Huh7 derived exosomes. Normalized for b-actin the DDct were indicated as fold of induction. **p<0.01 (f) Comparison of H19 expression in HUVECs after exosomes treatment. Real time-PCR analysis for H19 expression on HUVECs treated for 18 h with 5 mg/ml of SkHep or Huh7 exosomes. Normalized for b-actin the DDct were indicated as fold of induction respect to control (untreated cells). (DOCX 855 kb

Characterization of CB CD34+ cells grown in MH-CM culture.

<p>A- Growth curve of cells grown in MH-CM; a representative experiment out of 10 is shown. B- Percentage of adherent and non-adherent cells grown in MH-CM at day 30; a representative experiment out of 10 is shown. C- Phase-contrast morphology of day 30, adherent and non-adherent cells (original magnification 10x). Non-adherent cells are round and more refractive, while adherent cells are flat and less refractive; a representative experiment out of 10 is shown.</p

Phenotypic characterization of adherent cells in MH-CM culture.

<p>A- Immunophenotypic characterization of adherent cells generated by CD34+ cells cultured in MH-CM for 30 days. Mean values +/− SD of 15 independent experiments are shown. B- Flow cytometry analysis of the indicated endothelial markers expressed by adherent cells generated by CD34+ cells cultured in MH-CM for 30 days. Cells have been labelled with CD45, CD144 and with the indicated antigen. Shown are plots of CD45 negative cells (87+/−9%). The percentage of double-positive cells for the two endothelial antigens are given. A representative experiment out of 15 is shown. C- Confocal imaging: triple immunostaining analysis for the indicated antigens of adherent cells generated by CD34+ cultured in MH-CM for 30 days. A representative experiment out out of 5 is shown.</p