Supplementary MaterialsSupp Physique S1-S3. effects on a common liver stem cell

Supplementary MaterialsSupp Physique S1-S3. effects on a common liver stem cell populace. Assessment of potential mechanisms through which TGF- signaling may promote liver tumor formation in the setting of p53 loss revealed a subset of tumors that express increased levels of alpha-fetoprotein. Furthermore, tumors from mice express increased TGF-1 levels BAY 80-6946 inhibitor database compared to tumors from mice. Increased phosphorylated Smad3 and ERK1/2 expression was also detected in the tumors from mice and correlated with increased expression of the TGF- responsive genes, and are also common molecular events in human liver cancer (9). In addition to mutations, alterations in the transforming growth factor-beta (TGF-) signaling pathway are commonly observed in HCC. TGF- is usually a secreted cytokine that initiates downstream signals through binding to a heteromeric cell-surface receptor complex that consists of two transmembrane serine-threonine kinases, TGF- receptor, type I (TGFBR1) and type II (TGFBR2). This activated receptor complex induces both Smad-dependent and Smad-independent signaling pathways (10). TGF- has been found to be overexpressed in 40% of HCCs (11), while Tgfbr2 has been shown to be downregulated in 37-70% of tumors (12, 13). In the liver, TGF- has been shown to play both tumor suppressive and tumor promoting functions (14, 15). This paradoxical role of BAY 80-6946 inhibitor database TGF- in malignancy is usually believed to be a consequence of the context dependence of the TGF- signaling pathway on tumor cells. Among other factors, the concurrent gene alterations present in a tumor cell can influence whether TGF- signaling has primarily an oncogenic or tumor suppressive role. Thus, it is important to determine cooperative effects of specific gene mutations around the TGF- signaling pathway in order to BAY 80-6946 inhibitor database determine what effect therapies directed at the TGF- pathway may have on cancers transporting specific mutations that impact the pathway output (16). Studies from systems have revealed that p53 and TGF- can cooperate to regulate a number GCN5 of cellular responses (17). p53 actually interacts with Smad2 and Smad3 BAY 80-6946 inhibitor database in a TGF- dependent manner (18). In mouse embryonic fibroblasts, p53 is required for TGF- mediated growth arrest, and in promoter, p53 binding is required for expression and is believed to help stabilize a larger complex consisting of Smad2, Smad4, and FAST1 (18). Additionally, the repression of alpha-fetoprotein (HCC formation remains to be determined. Thus, we developed a mouse model system to investigate if p53 and Tgfbr2 cooperate to impact HCC formation. Materials and Methods Generation and characterization of Alb-Cre;Trp53flx/flx;Tgfbr2flx/flx mice The generation of (Alb-Cre), Trp53F2-10/F2-10 (Trp53flx/flx) and Tgfbr2flx/flx mice has been described previously (21-23). mice were crossed with transgenic mice and mice to generate the following genotypes: ((((Control). Mice were backcrossed in order to obtain a strain background that was on average C57BL6 (87.5%) / FVB (12.5%). Both male and female mice were used for this study. Tissues from non-breeders were utilized for qRT-PCR, ELISA and Western Blot assays. Genotypes were determined by PCR following published protocols (21, 24). All mice were managed and cared for using protocols approved by the institutional IACUC. Mice that became moribund or reached approximately 15 months of age were sacrificed and necropsied. Total body weight and liver excess weight were measured. Mouse tissue processing Mouse tissues were either snap-frozen in liquid nitrogen and utilized for RNA and protein preparations; or fixed in 10% neutral buffered formalin phosphate (Fisher Scientific, Pittsburgh, PA), embedded in paraffin, and slice into 4 m sections for H&E staining or immunohistochemistry (Observe Supporting Information). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) Gene expression studies were performed as explained in Supporting Information. The results of the qRT-PCR assays were normalized to -glucuronidase. Statistical analysis was performed using the GraphPad Prism version 4.00 software. The Mann-Whitney test was utilized for comparisons of quantitative results BAY 80-6946 inhibitor database from the ELISA and qRT-PCR assays. A value of 0.05 was regarded as significant. Protein lysate preparation Total protein lysates were prepared from frozen tumor or non-tumor liver tissue. Samples were homogenized on ice with a Dounce Tissue Grinder (Wheaton Science Products, Millville, NJ) in Triton X-100 Lysis Buffer (Observe Supporting Information). TGF-1 ELISA Mouse TGF-1 was assessed in protein lysates (21ug per sample) obtained from selected paired frozen tumor and non-tumor tissues, as well as from grossly normal appearing livers. The samples were activated and quantified according to the manufacturers instructions (R&D Systems, Minneapolis, MN). Western blot analysis Protein lysates (20 ug per lane) were resolved by 10% SDS-PAGE and transferred to PVDF membranes (Thermo Scientific, Rockford, IL). Antibodies used are explained in the Supporting Information. Densitometric quantification of immunoblots was performed using the ImageJ 1.43 software. Results Targeted deletion of.