Fig 1.
TGFbeta1 induces hepatocyte binucleation.
(A) Percentage of binucleated cells in cultures of MMH/E14 cells maintained in RPMI w/o additional growth factors for three weeks and then supplemented with the indicated cytokines and growth factors for 72h. (CTRL = untreated cells). (B) Immunofluorescence of untreated and TGFbeta1-treated MMH/E14 and WT/3A hepatocytes, and pleural mesothelial MeT-5A cell line after staining with phalloidin (red) and DAPI (blue). Images of MeT-5A cells was acquired by confocal microscopy. (C) Percentage of binucleated cells in cultures of hepatocyte cell lines (MMH/E14 and WT/3A) and of pleural mesothelial (MeT-5A) cell line, after 72 h of TGFbeta1 treatment; data are expressed as average values of four independent experiments, ± s.e.m.
Fig 2.
The in vivo inhibition of TGFbeta receptor hampers hepatocyte binucleation.
(A) Experimental schedule for in vivo 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).
Fig 3.
The hepatocyte binucleation results from a Src-dependent failure of cytokinesis.
(A) Time-lapse video microscopy of single untreated (upper) and TGFbeta1-treated (lower) MMH/E14 hepatocytes. The dynamics of mitosis are compared and the cytokinesis failure in a single TGFbeta1-treated cell is shown in the last frame of the lower panel. (B) Western Blot analysis of the phosphorylated form of AKT (upper panel), target of PI3K, in the presence of TGFbeta1 with or without LY294002 (PI3K inhibitor, 10μM). CDK4 and tubulin were utilized as loading control. (C) Western Blot analysis of the phosphorylated form of Src kinase (upper panel) in the presence of TGFbeta1 with or without PP2 (Src inhibitor, 2μM). Src activity was analysed as autophosphorylation. Tubulin was utilized as loading control. (D) Percentage of binucleated cells in TGFbeta1-treated or untreated cultures in the presence or absence of the PI3K inhibitor, LY294002, or the Src inhibitor, PP2. Data are expressed as average values of three different experiments ± s.e.m. (**p value < 0.01). (E) Proliferation assay performed by cell count after 72 h of culture in the shown conditions. Data are expressed as average values of three different experiments ± s.e.m., and plotted as ratio between treated and untreated (CTRL = 1) cells.
Fig 4.
The TGFbeta1-induced cytokinesis failure is associated with the delocalization of active RhoA from the midbody.
(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.
Fig 5.
Tetraploid hepatocytes proliferate maintaining the poliploidy state in their progeny after TGFbeta1 withdrawal.
(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.
Fig 6.
Graphical representation of the proposed TGFbeta-induced mechanism of hepatocyte binucleation.