The up regulation of ER by 4OHT contributes to anti oestrogen resistance in ER negative breast cancer cells ER positive breast cancers generally have a better prog nosis and are often responsive to anti oestrogen different therapy, which was the first example of a successful therapy targeting a specific protein, the ER. Unfortunately, ER negative breast cancers are more aggressive and un responsive to anti oestrogens. Although the transform ation of ER negative into ER positive cells by gene therapy or ER gene re expression are common strat egies to restore anti oestrogen responsiveness, we observed in this study that MDA MB 231 ER negative cells were intrinsically resistant to 4OHT despite the overexpression of ER.

Proliferation assays to determine the concentration and time dependent effects of 4OHT on MDA MB 231, showed that this drug stimulated cancer cell proliferation at concentrations as low as 1 nM. Stimulation of cancer cell proliferation, in the presence of 4OHT, was also observed in hormone deprived conditions, which indicated that this effect was independent of oestrogens in the culture medium. This class of resistance to tamoxifen was firstly discovered by Gottardis and Jordan in MCF7 cells and the results agree with additional observation in which long term tam oxifen treatment of MDA MB 231 cells increased their growth and their aggressiveness in animal tumours. ER positive tumours are more differentiated and have lower metastatic potential than ER negative tumours.

Because this suggests a protective role of the oestrogen receptor in tumour progression and metastasis, we next examined whether the 4OHT induced expression of ER resulted in a decreased metastatic potential of MDA MB 231. Using a wound healing assay, we observed that the treatment of MDA MB 231 with 4OHT greatly increased the migratory ability of these cells. These results indicated that the re expression of ER in ER negative breast cancer cells may promote cell growth and the resistance of these cells to anti oestrogen therapy. To determine whether ER plays a role in 4OHT resist ance, we examined the effect of a decreased level of ER on the growth of MDA MB 231 cells. Because the treat ment of these cells with U0126 clearly inhibited the ER expression induced by 4OHT, we first ana lysed whether this drug also inhibited 4OHT induced cell proliferation. As shown in this Figure, U0126 reduced the growth of cells that were treated with 4OHT and untreated cells to the same extent. These results indi cated that the MAPK cascade controls the proliferation of this breast cancer cell line. It is well known that mem brane ER plays a role in the temporal coordination of phosphorylation/dephosphorylation Brefeldin_A events for the ERKs in breast cancer cells.

aerogenes bacteria. The plating efficiency of cyst spores was 70%, similar to that of spores collected selleck from fruiting bodies on filters, which was 66%. Thus, terminal cell differentiation occurred in radially symmetrical fash ion in the absence of the normal morphogenetic move ments of culmination. This contrasts with the slug like elongated and linearly polarized aggregates formed when cells were agitated in high O2. The radially polar ized organization may result from a more uniform envir onment presented by the static setting in which polarizing gradients of O2 or NH3 fail to form. Under 21% O2, stalk cells and spores were rarely observed in the less compacted aggregates that form under these conditions. When present they occurred as clusters or single cells.

At 40% O2, larger aggregates were formed but they lacked dense cores observed at higher O2 levels. These cyst like aggregates possessed a stalk cell cortex but their interior cells pro duced few spores, as visualized after squashing. Though spores were not detected in this example, variable numbers were observed over the 5 in dependent trials as quantitated in Figure 4C. The vari ation suggests that 40% O2 is close to the threshold required for sporulation whose exact value is likely influ enced by other factors, as observed for culmination. To address the differentiation status of cells at the lower O2 levels, extracts were Western blotted for the spore coat precursor proteins SP85, SP96 and SP75 that are markers of prespore cell differentiation. Whereas all 3 glycoproteins appeared in Ax3 cells by 24 h at 70% O2, negligible expression occurred at 20% after 3 d.

Thus increasing O2 levels were required for tight aggregate formation, terminal stalk cell differenti ation, and differentiation of the interior prespore cells into spores. It is likely that metabolic O2 consumption results in intracyst hypoxia in these unstirred cultures which, in the submerged state, is not adequately replen ished by O2 diffusion. The finding that elevated O2 ten sion in the atmosphere above the medium can rescue terminal differentiation indicates that O2 availability is the limiting factor for terminal cell differentiation in this setting. It is not evident whether the higher O2 level required for spore compared to stalk cell differentiation reflects a higher O2 threshold requirement for spore dif ferentiation or lower O2 in the aggregate centers.

Requirement of PhyA for sporulation in submerged conditions A previously described mutant strain disrupted at its phyA locus GSK-3 was analyzed to determine the involve ment of Skp1 prolyl 4 hydroxylation in submerged de velopment. phyA cells formed cyst like structures at 40 100% O2 with outer layers of differentiated stalk cells, similar to the normal Ax3 strain.

Gene sets with 2 fold or more Veliparib FDA difference in mRNA level and p value cut off are presented in Table 1. Statistical analysis The effect of doxorubicin or NS398 on breast cancer cell proliferation was analyzed using one way ANOVA followed by Turkeys multiple test. The data of in vitro cancer cell invasion and tumor incidence in the mice were analyzed using Students t test. Results Resistance of MCF 7 DOX cells to doxorubicin Doxorubicin is one of the most commonly used drugs in the treatment of cancer, but its inhibitory effect on cell proliferation varies in several cancer cell lines. Therefore, we investigated whether doxorubicin has the same anti proliferative effect in MDA MB 231, MCF 7, MCF 7 DOX, and T 47D cell lines. The cells were treated with the indicated concentrations of doxorubicin for 72 h and cell proliferation was measured using the MTT assay.

Doxorubicin inhibited cell proliferation in a concentra tion dependent manner in MCF 7 and T47D cells, and to a lesser extent in MDA MB 231 cells. By contrast, doxorubicin mediated inhibition was significantly reduced in MCF 7 DOX cells. We next mea sured the growth of MCF 7 and MCF 7 DOX cells at lower doxorubicin concentrations and MCF 7 DOX cells were consistently resistant to doxorubicin. We then tested whether the doxorubicin mediated growth inhibition was mediated by apoptosis. After MCF 7 and MCF 7 DOX cells were treated with 1 uM doxorubicin for 48 h, terminal deoxynucleotidyl trans ferase mediated dUTP nick end labeling based fluores cence activated cell sorting analysis showed that doxorubicin did not induce apoptosis in MCF 7 DOX cells.

however, doxorubicin did induce apoptosis in MCF 7 cells. We further confirmed the resistance of MCF 7 DOX cells to doxorubicin by Wes tern blot analysis. Induction of PARP cleavage in MCF 7 cells confirmed that doxorubicin induced apoptosis in these cells. However, PARP was not cleaved in MCF 7 DOX cells treated with doxorubi cin. Acquisition of invasive and metastatic properties in MCF 7 DOX cells Intrinsic or acquired drug resistance results in disease recurrence and metastasis. We analyzed changes in gene expression in doxorubicin resistant MCF 7 DOX cells using DNA array analysis. Differentially expressed genes related with invasion are listed in Table 1. We next examined whether MCF 7 DOX cells acquired metastatic properties.

First, we measured the enzymatic activities of MMP 9, MMP 2, and uPA in MCF 7 and MCF 7 DOX cells by gelatin and fibrino gen plasminogen zymography. The enzymatic activities of MMP 2, MMP 9, and uPA were markedly higher in MCF 7 DOX cells than in non invasive MCF 7 cells. Increased levels of MMP 9 and MMP 2 expression have been correlated with an invasive pheno type of cancer Batimastat cells. Thus, we assessed the invasive ness of MCF 7 and MCF 7 DOX cells using in vitro invasion assays. As expected, the MCF 7 DOX cells were significantly more invasive than MCF 7 cells.

Additionally, we observed that exposure of H9c2 cells to the either pan HDAC inhibitor affected neither the expression nor sub cellular distribution Gemcitabine IC50 of HDACs or sir tuins. Pan HDAC inhibitors alter global gene expression profiles of H9c2 cells The main aim of our study was to examine the effect of HDACIs on gene expression in cardiac myocytes with out other cell types that coexist in the intact heart. We serum starved H9c2 cells for 16 24h before initiating drug treatment by incubating the cells in complete growth medium and growth media supplemented with CBHA or TSA. Based on our empirical assessment of the actions of HDACIs in cell cycle synchronized H9c2 cells, in the presence or absence of IL 18, we believe that 6h and 24h time points of treatment will yield snap shots of genome wide actions of CBHA and TSA during early and late stages of cell cycle.

Messenger RNAs extracted from six replicates of each treatment cohort were processed for hybridization to Illumina rat micro arrays and subsequent analysis. We filtered the gene ex pression dataset through the criteria of absolute 2 fold change and p value of 0. 01 before analyzing these data by principal component analysis and the un supervised hierarchical clustering methods. As shown in Figures 2 and 3, the cohorts of vehicle treated H9c2 cells harvested at 6h and 24h oc cupy close, albeit unique positions in the PCA graph. Similarly, the replicates of CBHA or TSA treated cells harvested at 6h and 24 h after treatment are also uniquely grouped in the PCA graph. The RatRef 12 Expression BeadChip contains about 21,900 genes.

Exposure of H9c2 cells to TSA and CBHA led to a total of 672 and 1485 differentially expressed genes, respectively. It appears therefore that the expression of approximately 3% and 6% of genes were significantly affected in H9c2 cells in response to TSA and CBHA, respectively. Based on their temporal expression characteristics and quantification of their expression levels, the TSA and CBHA responsive genes could be organized into six distinct clusters, A through F. The sizes of Clusters C and F elicited in TSA treated cells were much larger compared with their counterpart clusters in CBHA treated cells. This is in contrast to what occurred in H9c2 cells treated with CBHA that induced more nu merous transcripts belonging to Clusters B, D and E.

As depicted in Figure 4, TSA elicited differential ex pression of 468 and 231 genes at 6h and 24h post treat ment, respectively. An identical exposure AV-951 of H9c2 cells to CBHA for 6h and 24h elicited 768 and 999 DEGs, respectively. Ingenuity pathway analysis indicates that CBHA and TSA perturb overlapping yet distinct gene networks in H9c2 cardiac myocytes We began our gene network studies with the reasoning that interrogation of the maximum numbers of DEGs by IPA would reveal the most robust networks involved in the actions of TSA or CBHA.

Additionally, increasing read this the PCC threshold resulted in fragmentation of networks into a large number of structured subgraphs, reflected in the number of connected components and clustering coefficients. Overall, networks derived from hypertrophic tissues were highly structured, characterized by nodes with multiple connections, small network diameters and relatively high clustering coefficients. Co expression model of Physiological cardiac hypertrophy Due to the large number of genes and co expression links observed in this analysis, some observations could be due to experimental artifacts and thus of questionable biologi cal relevance. The recurrence of a co expression link in all three microarray datasets was considered to increase the reliability of the inference. At PCC 0.

70, the Akt and PI3K networks shared 6990 genes and 70347 interactions, the PI3K and Swimming networks shared 5709 genes and 77718 interactions, and the Akt and Swimming networks shared 4521 genes and 34250 interactions. There were 2128 genes and 4144 interactions common to all three networks, which formed a consensus Conserved gene co expression network. Similarly to the Akt, PI3K, and Swimming networks, the Conserved network was scale free. To evaluate the statistical significance of the Con served network, three randomized networks were gener ated. Randomization was performed by shuffling edges of the Akt, PI3K, and Swimming networks 4�� times, while preserving the node degrees of the original networks This procedure was repeated 200 times.

The simulation showed that on average, the three random networks shared 1519 co expressed genes and that at most their intersection contained 1641 genes. These results indicated that identification of 2128 genes in the Conserved network is statistically signifi cant. Phenotype specificity of the Conserved network was estimated by comparing it to gene co expressions inferred from the Normal mouse transcriptome. Drug_discovery It was hypothesized that the extent of conserved nodes and edges between two networks may correspond to mole cular mechanisms shared by the LVH phenotype and cells under basal conditions. Interestingly, it was deter mined that the Conserved and Normal networks shared only 88 genes and 57 co expressions, confirming that the Conserved network may reflect LVH specific cardiac response. To gauge the extent of validated molecular pathways in all co expression networks, all genes were mapped to the KEGG pathway database. Genes with annota tions in KEGG pathways were considered to be true positives and network precision was esti mated as the proportion of true positive genes to the overall number of genes in a network. At PCC 0. 70, net work precision for the Akt, PI3K, Swimming, and Con served networks approached 31%.

Non specific binding of antibodies to dying cells was ruled out in every e periment since anti DCs markers binding to isolated Apo Nec cells was less than 5% or absent. DCs maturation was also evaluated by the decrease of DCs endocytosis after Apo Nec cells phagocytosis. FITC D endocytosis was ma imal in iDCS, 99 5% uptake, decreased to 31 3% in LPS maturated DCs and to 33 5% in DC promotion info Apo Nec cells. DCs loaded with melanoma apoptotic necrotic cells e press CCR7 and migrate towards MIP 3 beta in vitro DCs migration to lymph nodes is crucial to trigger T lym phocyte priming. MIP 3 chemokine drives the homing of mDCs to the lymph node and interacts specifically with its receptor CCR7 e pressed on the cell surface. iDCs e pressed low levels of CCR7 but increased its e pression after Apo Nec phagocytosis or LPS maturation.

We observed that iDC migrated to MIP 1 but did not respond to MIP 3, on the contrary DC Apo Nec and DC LPS migrated to MIP 3 but failed to respond to MIP 1. Thus, DC Apo Nec cells e press MIP 3 receptor CCR7 and are able to migrate in response to MIP 3, potentially allowing their homing to lymph nodes. DCs loaded with apoptotic necrotic melanoma cells cross present MelanA MART 1 and gp100 Ags to specific T CD8 cells An important issue in this work was to assess if melanoma associated Ags present in the Apo Nec cells mi ture could be cross presented to specific CTLs after DCs phagocytosis. We analyzed IFN secretion Dacomitinib by specific CD8 T clones for MelanA MART 1 and gp100 after overnight stimula tion with DC Apo Nec cells.

As observed in Figure 6A and 6B, DC Apo Nec co cultured with the CTL clones effi ciently induced IFN secretion as soon as 6 hs and up to 48 hs after phagocytosis, evidencing cross presentation for both Ags. For MelanA MART 1 Ag we also observed IFN secretion after incubation with DCs loaded with Apo selleckchem Nec HLA A 0201 negative MelanA MART 1 cells clearly demonstrating cross presenta tion for this Ag. Controls for this e periment were performed, showing specific HLA A 0201 restricted response using either DCs loaded with the corresponding peptides or CTL stimulation with live HLA A 0201 posi tive gp100 MelanA MART 1 melanoma cells but lack of response using live MEL Y2 cell line or with MelanA MART 1 peptide for G154 clone or gp100 peptide for M27 clone. Mel Y3 was rendered Apo Nec by irra diation and also assayed for CTL priming, however, it only induced IFN secretion by the CTL clones at short times after irradiation and culture. After 48 72 hs of culture, when HLA A 0201 positive Apo Nec cells have completed the apoptotic necrotic process, they failed to present both Ags to CTLs.

Previous data have already shown that high levels of p130Cas correlate with intrinsic resistance to tamo ifen treatment in a large subset of estrogen inhibitor expert receptor positive human breast tumors. Moreover, in human breast cancers overe pression of both HER2 and p130Cas is associated with poor prognosis. Conclusions Overall in this work we demonstrate the involvement of p130Cas in mesenchymal breast cancer cell plasticity, highlighting a new pathway linking p130Cas to Co 2 through c Src and JNK activities. p130Cas is thus emerging as a critical player for onset and progres sion of many aggressive cancers, strengthening its rele vance as an unfavorable prognostic marker and a putative therapeutic target, mostly in combination with high levels of ER, HER2 or Co 2, respectively.

Introduction Rheumatoid arthritis is characterized by inflamed synovial tissue containing a massive infiltration of lym phocytes and macrophages with synovial fibroblast prolif eration. IL 18, an IL 1 family member, is involved in RA pathogenesis. We and others have shown that IL 18 plays an important role in the immune response, in local or systemic angiogenesis, and in monocyte recruit ment. Various sources of IL 18 have been identified in cluding antigen presenting cells, as well as keratinocytes, articular chondrocytes, osteoblasts, and synovial fibro blasts. IL 18, is produced as a biologically inactive precursor protein containing a propeptide domain localized to the cytoplasm. To be activated, pro IL 18 requires cleavage by the IL 1B converting enzyme, which is a member of the aspartate specific cysteine protease family.

Caspase 1 is pro duced as an inactive form. To be activated, its needs to be cleaved into 20 kDa and 10 kDa subunits. Both sub units form heterodimers with interactions with other proteins and are involved in inflammasome formation and activation of inflammatory processes. Active caspase 1 is located in the plasma membrane, where it cleaves pro IL 18 to IL 18. Caspase 1 and pro IL 18 IL 18 are comple ed to other proteins that are involved Entinostat in the secretion of IL 18. Caspase 1 is also a critical putative target in patients with cryopyrin associated periodic syndromes. When IL 18 is secreted, it becomes active. IL 18 bioactivity is dependent on both IL 18 and IL 18 binding protein levels. Among various signaling pathways, the mitogen activated protein kinase family, nuclear factor kappa light chain enhancer of activated B cells and janus activated kinase pathways are thought to be critical in RA pathogenesis. All these pathways can be activated by TNF. We previously des cribed ways to research use only regulate TNF induced IL 18 bioactivity in RA synovial fibroblasts by modulation of IL 18 or IL 1BP.

Hence, the activation of p38 and Akt pathways upon infection appears to be either non essential for HAstV1 infection or redundant with other pathways that could relay the essential signals for the infectious processes. It is interesting to note that wortmannin treatment showed no blockade of RNA replication, but e hibited a block in viral release. Immunofluorescent detection of viral capsid protein revealed that treatment with wortmannin caused unusual punctate staining of the capsid protein, which suggests that the reagent failed to block viral entry, but was effective in delaying the process leading to capsid e pression showing aberrant distribution.

The time point e amined for viral RNA replication, 24 hpi, may have been the point when viral RNA replication had already reached a plateau, but the inhibitory effect of wortmannin on the release of RNA and virion may have been visible because of the delay of the infectious process. Treatment with triciribine enhanced viral RNA replica tion in HastV1 infected cells, which possibly caused the increased viral release that was inferred from the level of viral RNA and capsid protein in the culture supernatant. Surprisingly, we found that the Akt phosphor ylation was not effectively blocked at 24 hpi and viral capsid release was enhanced in a dose dependent manner. We also noted that triciribine treatment slightly enhanced cell viability. Overall, the treatment appeared to have a positive effect on viral propagation in our e periments, rather than an inhibitory effect. Similarly, treatment with NSC23766 or Y27632 increased the e tent of viral RNA replication.

Interestingly, a marked increase in the phos phorylated Akt level was observed in cells treated with each drug. Akt activation is known to involve a feedback loop activating Rac1, led by ROCK inhibition using Y27632. Because Rho family sig Cilengitide naling events are known to involve balanced regulation, inhibition of another member of the Rho family, Rac1, by NSC23766 could also have activated such a feedback loop. The activated Akt possibly caused an in crease in protein synthesis, which could enhance viral RNA replication. We noted that two Akt phosphorylation inhibitors affect HAstV1 infection differently. Triciribine apparently increased the amount of viral RNA and the release of viral RNA and capsid in the culture supernatant, whereas MK2206 did not.

This difference could be due to a difference in the drugs inhibitory mechanisms. Triciribine inhibits Akt phosphorylation by binding to the PH domain of Akt, thereby blocking its recruitment to the plasma membrane, whereas MK2206 binds to the catalytic domain of Akt and inhibits its phosphor ylation. Triciribine is also known to inhibit cellular DNA synthesis. Nonetheless, neither Akt inhibitor blocked viral infection. In summary, our study has revealed that two signaling pathways, mediated by ERK and PI3K, are important for HAstV1 infection.

In the current studies we analyzed a panel of human melanoma cell lines with defined oncogenic alterations for sensitivity to PL 4032. In addition, with a view to development of a biomarker to indicate response to tar geted therapy, we investigated a non invasive method of imaging resistance versus sensitivity in vivo. We describe that PL 4032 works differentially in melanoma cell lines with BRAFV600E mutations and that the positron emission tomography tracer 2 fluoro 2 deo y D glucose can be used in non invasive PET imaging to dis tinguish between sensitive and resistant cell lines. Materials and methods Reagents and cell lines PL 4032 was obtained under a materials transfer agreement with Ple ikon and dissolved in DMSO to a stock concentra tion of 10 mM.

SKMEL28 was obtained from American Type Culture Collection, and the remaining human melanoma cell lines were established from patients biopsies under UCLA IRB approval 02 08 067. Cells were cultured in RPMI 1640 with L glutamine con taining 10% fetal bovine serum and 1% penicillin, streptomycin, and amphotericin. All cell lines were mycoplasma free when periodically tested using a Myco alert assay. BRAFV600E mutation analysis Genomic DNA was e tracted using Fle iGene DNA Kit and the 200 bp region flanking the mutation site was amplified by PCR using Invitrogen online primer design as described. The PCR products were purified using QIAquick PCR Purification Kit, sequenced and aligned with the BRAF gene. Oncomap 3 core mass spectrometric genotyping Samples were run through OncoMap 3 which interro gates 396 somatic mutations across 33 genes.

Whole genome amplified DNA at 5 ng ul was used as input for multiple PCR as described previously. Single base pair primer e tension was performed in a 2 ul reaction volume using iPLE Gold single base e tension enzyme. Products were res ined and transferred to SpectroCHIPs for analysis by MALDI TOF mass spectrometry. All mutations were confirmed by direct Batimastat sequencing of the relevant gene fragment. SNP array analysis DNA e tracted from the full panel of 13 human mela noma cell lines was hybridized onto Illumina Beadchip Human E on 510S Duo. DNA copy number was calculated using PennCNV as described. Eight of the cell lines were additionally ana lyzed using Affymetri GeneChip Human Mapping 250K Nsp Array.

Cell proliferation and viability assays Melanoma cell lines were treated in triplicates with PL 4032 and parallel vehicle control in the given concen trations for 120 hours. Viable cells was measured using a tetrazolium compound T}, where C1 the ini tial cell number, C2 the final cell number, and T 24 hours. The average of day 3, 4, 5 was used as the optimal doubling time for the given e perimental condition. Phosphoflow staining Cells were plated and treated with 1 uM PL 4032 or vehicle control for 1 or 20 hours, fi ed in 1.

Conversely, Vav3 e pression was unaffected by doceta el treatment. To determine the doceta el sensitivity of si Vav3 treated cells, cells transfected with si Vav3 or si Scr were treated with 5 nM doceta el for 72 h and assayed for cell prolifer ation and live death analyses. Treatment with doceta el or si Vav3 inhibited cell growth in a time dependent manner, and when LNCaPH cells were treated with si Vav3 in the presence of doceta el, sensitivity to doceta el was signifi cantly enhanced. We further con firmed this enhanced cell growth inhibition with the results of the cell live death assay. The assay stains live cells with a green fluorescence dye and dead cells with a red fluorescence dye. We observed that control si Scr and three independent e periments.

These results suggest that LNCaPH cells display Akt and ERK activation and that si Vav3 negatively regulates PI3K Akt and ERK pathway activation, enhancing the effects of doceta el. Effects of si Vav3 and doceta el on the apoptotic cell death of LNCaPH cells To investigate whether the growth inhibitory effects of the combination of si Vav3 and doceta el may be triggered by increased apoptosis in LNCaPH cells, we evaluated the apoptotic cells by flow cytometry, which assessed a sub G1 population of apoptotic cells, and enzyme linked im munosorbent assay using Cell Death Detection ELISAPLUS. Treatment with 5 nM doceta el led to in creased apoptosis in LNCaPH cells in a time dependent manner, but the sub G1 population was slightly increased by si Vav3 alone. When LNCaPH cells were treated with si Vav3 plus doceta el, a strong induction of apoptosis was observed.

Similarly, the addition of si Vav3 to doceta el markedly induced apoptosis in a doceta el concentration dependent manner. Among cells treated with si Vav3 plus 5 nM doceta el for 72 h, 42. 4, 9. 0, 10. 8, and 37. 8% of cells were in the sub G1, G1, S, and G2 M fractions, respectively. In LNCaPH cells treated with si Vav3 or 5 nM doceta el for 24 h, 7. 3 and 19. 6 fold increases in DNA fragmentation, respectively, were recorded, but combination treatment resulted in a 40. 2 fold increase in DNA fragmentation compared with the untreated control. These results are consistent with the significant growth inhib ition of LNCaPH cells induced by si Vav3 plus doceta el, and these combined effects were associated with Carfilzomib a large increase in the number of apoptotic cells. Because apoptosis can be triggered by death receptor mediated or mitochondria mediated cascades depend ing on the type of caspase activation, we evaluated caspase 8, caspase 9, and caspase 3 activation and sub sequent cleavage of PARP engaged in DNA repair in LNCaPH cells treated with si Vav3, 5 nM doceta el, or si Vav3 plus 5 nM doceta el for 48 h.