1. Volume 39, Issue 3, Pages 374-382 (September 2003)
Trichostatin A induces differential cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes 
Peggy Papeleu, Pascal Loyer, Tamara Vanhaecke, Greetje Elaut, Albert Geerts, Christiane Guguen-Guillouzo, Vera Rogiers 
Journal of Hepatology 
Volume 39, Issue 3, Pages (September 2003)
DOI: /S (03)

2. Fig. 1
Effect of TSA on DNA synthesis and histone acetylation in primary hepatocytes. Hepatocytes were cultured in triplicate at a density of 0.4×105 cells/cm2. Cells were harvested at the indicated time points. Six hours prior to harvesting, the cells were treated with [methyl-3H]-thymidine (2μCi/ml medium). (A) Dose-dependent inhibition of DNA synthesis. Hepatocytes were cultured in the presence of EGF and exposed to different concentrations of TSA: 0.1, 0.5 and 1 μM. TSA treatment started 4 h after plating and was maintained during the whole culture period (n=4). (B) Time-dependent inhibition of DNA synthesis. TSA treatment (1 μM) started either at time of plating (T0) or after a 4h-culture period (T4) and was maintained during the whole culture period (n=4). (C) Histones were extracted at the indicated time points and their acetylation status was evaluated by Western blotting (n=3).
Journal of Hepatology  , DOI: ( /S (03) )

3. Fig. 2
Dependence of growth arrest on the presence of TSA. (A) Hepatocytes were cultured in non-proliferative (−EGF/−TSA) or in proliferative (+EGF/−TSA) conditions. EGF-stimulated cultures received treatment with 1 μM TSA from time of plating until 24 or 60 h of culture. Six hours prior to harvesting, the cells were treated with [methyl-3H]-thymidine (2μCi/ml medium) and DNA synthesis was measured (n=4). (B) Hepatocytes were cultured in non-proliferative (−EGF/−TSA) or in proliferative (+EGF/−TSA) conditions. EGF-stimulated cultures received treatment with 1 μM TSA from time of plating until 24 or 72 h of culture. Expression of c-myc RNA as a function of culture time was analyzed by Northern blotting. 18S and 28S ribosomal RNA (methylene blue staining) are shown as a control of equal sample loading and sample integrity. (C) Hepatocytes were cultured in non-proliferative (−EGF/−TSA) or in proliferative (+EGF/−TSA) conditions. EGF-stimulated cultures received treatment with 1 μM TSA from time of plating until 24 or 72 h of culture. A representative Northern blot of cyclin D1 RNA expression is shown.
Journal of Hepatology  , DOI: ( /S (03) )

4. Fig. 3
Effects of TSA on late G1 and G1/S transition regulating proteins in primary hepatocytes. (A) Hepatocytes were cultured in the following conditions: −EGF/−TSA, +EGF/−TSA or +EGF/+TSA. TSA treatment (1 μM) started at time of plating and was maintained during 72 h of culture. At the indicated time points, total cell extracts were prepared from cultured hepatocytes. Proteins (50 μg) were separated on a 10% SDS-PAGE (n=3). (B) Equal sample loading was verified with Ponceau S. Histone H1 kinase activities were studied after purification of CDK 1 and 2 proteins on p9CKShs1 beads (n=3). (C) Immunoprecipitation of CDK2 proteins in total cell extracts. Histone H1 kinase activities of the immunoprecipitated CDK2 were determined as described in Section 2. (NIS: non-immune serum; IP CDK2: specific immunoprecipitation for CDK2) (n=3).
Journal of Hepatology  , DOI: ( /S (03) )

5. Fig. 4
TSA modulates the expression of immediate-early genes that regulate cell cycle entry. Liver was perfused either in the absence (A); or in the presence (B) of TSA (1 μM). Total RNA was extracted after dissociation of the cells from the liver (lane 1), decantation of the supernatants from the cells (lane 2), the first wash in Hepes buffer (lane 3), the second wash in antibiotics-supplied culture medium (lane 4) and at time of plating (lane 5) and was analyzed by Northern blotting. 28S ribosomal RNA is shown as a control of equal sample loading. For protein analysis, cell extracts were prepared at the indicated time points and proteins (50 μg) were electrophoresed and blotted as described in Section 2. (C) Histone H3 and H4 acetylation status in total liver, perfused either in the absence or the presence of TSA (1 μM). Blots shown are representative of at least three independent experiments. (D) Liver was perfused in the absence or in presence (E) of 1 μM TSA. Thereafter, hepatocytes were further cultured either under non-proliferative (−EGF/−TSA) or under proliferative, control conditions (+EGF/−TSA). TSA treatment (1 μM) continued at time of plating and was maintained during the whole culture period. Blots shown are representative of at least three independent experiments.
Journal of Hepatology  , DOI: ( /S (03) )

6. Fig. 5
Restoration of DNA replication depends on length of TSA exposure in G1. Liver was perfused in the continuous presence of 1 μM TSA and cells were subsequently cultured in the presence of EGF and TSA. TSA treatment was maintained until the time points indicated. DNA synthesis was measured by means of [methyl-3H]-thymidine incorporation at 72 h of culture. Results are expressed as % inhibition of the DNA synthesis of untreated cells and are the means±SD from three independent experiments.
Journal of Hepatology  , DOI: ( /S (03) )

7. Fig. 6
Effects of TSA on the viability of normal adult hepatocytes. (A) LDH leakage into the culture medium was measured at the indicated time points. *Significantly different results from values obtained in EGF-deprived medium (Paired Student's t-test, P<0.05; n=4). (B) DEVD-AMC caspase 3-like activities were measured at 48 and 72 h of culture. Results are presented in arbitrary units of fluorescence (per 100 μg total protein). **Significantly different results from values obtained in EGF-deprived medium (Paired Student's t-test, P<0.001; n=3) (C) Representative Western blot for caspase-3 (n=3). (D) Representative Western blot for BclxL (n=3). Hepatocytes were cultured in the presence of EGF (50 ng/ml) and treated or not with 1 μM TSA from time of plating during 48 h. BclxL expression was analyzed at 48 h of culture.
Journal of Hepatology  , DOI: ( /S (03) )

8. Fig. 7
Effects of TSA on hepatocyte function. Hepatocytes were cultured in the presence of EGF (50 ng/ml) and treated from time of plating until the indicated time points with 1 μM TSA. Albumin secretion into the culture medium was measured between 24–48 and 48–96 h of culture, respectively. Results are expressed as percentage of untreated EGF-stimulated cells and are the means±SD from four independent experiments. *Significantly different results from values obtained in EGF-stimulated untreated cells (Paired Student's t-test, P<0.05).
Journal of Hepatology  , DOI: ( /S (03) )