The percentage of the input DNA that was bound was calculated with the cycle threshold difference methodology and was averaged Inhibitor Library chemical structure over at least three experiments. Primers and reverse-transcription PCR determinations of RNA expression were as previously described.4 Briefly, total RNA from the homogenized mouse liver was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Complementary DNA was obtained by the reverse transcription of 2 μg of RNA with the SuperScript system (Invitrogen). Real-time PCR primers for indicated genes are listed in Supporting Table 5. Immunoblotting of whole cell and nuclear lysates was performed with the standard sodium
dodecyl sulfate–polyacrylamide gel electrophoresis methodology. Nuclear extracts from liver tissue, collected 2 days after the sham or PH surgery, were prepared as described.5 The primary antibodies were p53 (Santa Cruz), TA-p73 (Santa Cruz), HA tag (Cell Signaling), FoxO3 (Cell Signaling), and β-actin (Santa Cruz). Results are expressed as means and standard errors of the mean. Statistical analyses were performed with a t test; P values < 0.05 were considered
significant. We performed ChIP of liver tissue from 2-month-old WT mice with antibody against TA-p73 and isolated TA-p73–bound chromatin fragments for ChIP/chip. We uncovered 158 genes among the arrayed promoters as potential targets of TA-p73 binding activity in the liver (Supporting Table 1). In a separate CX-4945 in vivo set of experiments, we determined genes that changed expression after PH. Functional annotations of genes among the 158 TA-p73 target genes (Fig. 1A) and those that changed expression in response to PH (Fig. 1B,C) were performed with IPA. The analysis yielded 12 categories of
function, with cancer, cell death, and cell proliferation receiving the highest number of hits with the lowest P value, as determined selleckchem by IPA (Fig. 1). As expected, TA-p73–bound genes in the quiescent liver also included development, inflammatory response, cell signaling, and cell cycle categories (Fig. 1A).18, 19 Gene ontology analysis using DAVID and PANTHER bioinformatics tools yielded similar categories and additional ones, such as sensory perception and transcription (Supporting Figs. 1A and 2A). None of these gene targets had been previously reported as p73-regulated, and eight are known targets of p53 [e.g., annexin A1,20 Notch homolog 1 translocation-associated,21 and brain-specific angiogenesis inhibitor 122; Supporting Table 1]. Thus, many of the TA-p73 target genes identified by ChIP/chip are not known to be p53/p73-regulated and potentially define a specific set of TA-p73–regulated genes in the quiescent liver. Two-thirds PH promotes proliferation of liver cells and rapid growth of the remaining liver tissue and results in complete restoration of the organ mass in approximately 7 days after PH in mice. We collected liver tissue from mice 0, 0.