In the control group of the same genotype (TRRAPf/ΔCre+), only PBS was injected intraperitoneally. As another control, TRRAPf/Δ mice lacking Cre (TRRAPf/ΔCre−) were injected with pIpC. All the mice were maintained as approved by the Animal Care and Use Committee of the International Agency for Research on Cancer (Lyon, France) (ACUC 03/4). Mice were divided into three groups: Group 1 = TRRAPf/ΔCre+ www.selleckchem.com/products/BIBW2992.html without pIpC injection; Group 2 = TRRAPf/ΔCre+ with pIpC (to delete TRRAP); and Group 3 = TRRAPf/ΔCre− with pIpC (to compare the effect of pIpC alone).

At least three mice per group were examined per timepoint. Mice were fed a commercial diet and were given water adlibitum. To induce hepatic injury, 10 μL/g body weight of 10% solution of CCl4 in olive oil or olive oil alone was injected intraperitoneally 48 hours after the last pIpC injection, and the mice were sacrificed at the times indicated. Selleck C59 wnt Forty-eight hours after the third injection of pIpC was considered 0 hour for the collection of samples after CCl4 treatment. Mice were sacrificed and part of the liver was removed and fixed

in 4% paraformaldehyde for paraffin-embedded sectioning. Other parts of the liver were removed and frozen in liquid nitrogen and kept at −80°C for preparation of protein lysates and total RNA. Histologic and immunohistochemical analyses were performed after staining either with hematoxylin and eosin stain (H&E) or with Feulgen-solution (Schiff’s base) or were left unstained for marker analysis. 5-Bromo-2-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA) staining was performed as described,17 using specific antibodies (Supporting Table 1) and Vectastatin ABC alkaline phosphatase kit or ABC peroxidase kit (Vector Laboratories). At least 5,000 cells were scored for BrdU and PCNA index. Western blot analysis of liver nuclear proteins was carried out as described,13 using specific antibodies (Supporting Table 1). The ChIP assay was performed as described18 and Tangeritin according

to the manufacturer’s recommendations (Upstate Biotechnology, Lake Placid, NY), using polyclonal antibodies specific for histone modifications and transcription factors (Supporting Table 1). The recovered DNA was then analyzed by PCR using primers recognizing different regions of the cyclin A1 promoter (Supporting Table 2). For statistical analysis we used Student’s t test for comparison between groups. P-values <0.05 were considered statistically significant. To study the function of TRRAP and TRRAP-mediated histone acetylation in transcription and cell proliferation during tissue regeneration in response to acute liver injury, we used TRRAP-CKO mice that allow inducible deletion in vivo of the TRRAP gene in a spatiotemporal manner (Fig. 1A).