Relative protein expression levels were quantified by specific protein/GAPDH ratio, which are presented as mean ± standard deviation (SD) from three independent experiments (listed in Supporting Table 2). Cell proliferation was measure by a methyl

thiazol tetrazolium (MTT)-based proliferation assay, as described before.[10] Caspase-3/7 activity was determined using the Caspase-Glo 3/7 assay system (Promega, Madison, WI). Anchorage-independent soft-agar growth assay and quantitative reverse-transcriptase polymerase chain reaction (qPCR) was performed as previously described.[10] Cell lysates were incubated with indicated Daporinad molecular weight Staurosporine mw Abs and protein A/G beads

(Life Technologies Corporation, Carlsbad, CA) overnight. Immunoprecipitates were washed five times and then subjected to immunoblotting analysis. Luciferase reporter constructs containing the YAP promoter region were cloned into pGL3-based vectors, then stably cotransfected with a Renila luciferase expression plasmid into cells. Luciferase activities were analyzed using a dual-luciferase reporter kit (Promega). Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT express kit from Active Motif (Carlsbad, CA). Protein-DNA 上海皓元医药股份有限公司 complexes were incubated with 3 μg of anti-CREB Abs (#1496; Epitomics). HepG2 cells (5× 106) expressing shRNA or protein, as indicated, were subcutaneously (SC) injected into athymic nude mice (Bikai, Shanghai, China). Tumor size was measured every 6 days using a caliper, and tumor volume was calculated as 0.5 × L × W2, with

L indicating length and W indicating width. Mice were euthanized at 45 days after injection. We examined whether YAP and CREB were important for liver cancer cells. YAP- or CREB-specific shRNAs with high knockdown efficiency (Supporting Fig. 1) were used to silence expression in both Bel-7402 and HepG2 cells. We found that inhibition of either YAP or CREB decreased cell proliferation, compared to control, as measured by an MTT-based assay and Ki-67 immunostaining (Fig. 1A and data not shown). Furthermore, we found that both YAP and CREB knockdown impaired the ability of these cells to form colonies in soft agar (Fig. 1B), whereas they markedly increased apoptosis, as shown by increased caspase 3/7 activity and caspase 3 cleavage (Fig. 1C and data not shown).