An ANOVA analysis provided a few num ber of genes differentially regulated by lixisenatide versus placebo kinase inhibitor ARQ197 in the infarct area. Interestingly, lixisenatide was equipotent to GLP 1 and other GLP 1 receptor analogues on reducing infarct size in perfused isolated rat hearts. In contrast, lixisenatide at higher concentrations, but not GLP 1, improved frac tional shortening in isolated cardiomyocytes. This might suggest that lixisenatide displays acute cardioprotection via the GLP 1 receptor, while effects of lixisenatide on contractility are mediated by a different signalling path way. In line with our findings, Vila Petroff et al. showed the lack of contractility promoting effect of GLP 1 in isolated cardiomyocytes.

We supplemented their findings by demonstrating that even very high concentra tions of the GLP 1 peptide, not amenable to rapid degrad ation by dipeptidyl peptidase IV, were ineffective. Finally, when performing Inhibitors,Modulators,Libraries a similar set of experiments on cardiomyocytes from GLP 1 receptor knockout mice, a robust response to lixisenatide remained suggesting a GLP 1 receptor independent effect lixisenatide on the contractility response. Lixisenatide displayed beneficial effects in our rodent models, but GLP 1 receptor mRNA was not detectable without ambiguity. Two different PCR Taqman probes, located to different parts of the rat gene, did not reveal abundant expression of the GLP 1 receptor in rat hearts and cardiomyocytes, but indicated a strong expression in other organs like rat pancreas. Overall, controversial data on the expression of the GLP 1 receptor in mam malian cardiac tissues exists.

GLP 1 receptor mRNA expression in human heart samples was detected by an RNA protection assay. In contrast, using autora diography with a radio labeled GLP 1, no staining was detected in human heart despite several other organs be ing positive for this ligand. Inhibitors,Modulators,Libraries An antibody against the GLP 1 receptor stained in mouse heart several cell Inhibitors,Modulators,Libraries types. Later it was shown that available antibodies against the GLP 1 receptor display strong cross reactivity in cells not expressing the GLP 1 receptor. Hence, immunostainings should be carefully considered as long as specificity is not clearly demonstrated. Simultaneously to immunostainings, Ban et al. detected mRNA for the GLP1 receptor, using an endpoint PCR with a high number of amplification cycles followed by specific hybridization.

This methodology does not rule out a very low expression level in the samples. Bullock and co workers used the RNAse protection assay, a more quan titative but less Inhibitors,Modulators,Libraries sensitive methodology, to assess mRNA distribution in rat tissues for the GLP 1 receptor. They could not confirm Inhibitors,Modulators,Libraries expression in the rat heart www.selleckchem.com/products/Rapamycin.html in contrast to other positive tissues like pancreatic tissues. Currently, we cannot exclude that a GLP1R variant is expressed in the heart which is not detectable using the two different PCR primer assays.

Meanwhile, in CP4715 treated pellets, the expression of type II collagen Palbociclib Phase 3 and aggrecan was signifi cantly increased, whereas the expression of type I and type III procollagen was not suppressed, or rather enhanced, probably due to the preference in integrin inhibition Inhibitors,Modulators,Libraries of this compound. Although the echistatin Inhibitors,Modulators,Libraries treated pellets contained fewer cells than the other pellets, proteoglycan syn thesis was the greatest with those pellets, which was, again, consistent with the results of histological evaluation and gene expression analysis. weeks, and investigated whether any changes occurred in gene expression or matrix synthesis by the presence of echistatin in the media.

In this experiment, some pellets were cultured in the media containing CP4715, a synthetic Discussion The results of this study indicated that 5B1 integrin could play a pivotal role in the induction of noncartilaginous procollagen expression in dedifferentiating chondrocytes. Inhibitors,Modulators,Libraries Previous studies have reported various roles of 5B1 integrin in chondrocytes. 5B1 integrin could be a me chanoreceptor for chondrocytes, and may regulate proliferation and survival of the cells. 5B1 integ rin may also promote catabolic responses in chondrocytes, inducing the expression of matrix metalloproteinases and proinflammatory Inhibitors,Modulators,Libraries cytokines. Reactive oxygen species may be generated in chondrocytes upon the activation of 5B1 integrin. In those catabolic responses, ERK, p38 mitogen activated protein kinase, c Jun N terminal kinases, and protein kinase C pathways may be activated by this integrin.

Our current investigation has revealed another role of 5B1 integrin in articular chondrocytes to induce the Inhibitors,Modulators,Libraries expression of type I and type III procollagen. AKT signaling was considered to be involved in the induction. Although not known with chondrocytes, in fibroblasts, AKT signaling has been shown to induce the expression of type I procollagen. With the progression of de differentiation, chondrocytes come to present a fibroblast like phenotype. One might therefore reasonably consider that this reported role of AKT signaling in fibroblasts is acquired by cultured chondrocytes with the progression of dedifferentiation. Current finding might explain a phenotypic change of chondrocytes observed in vivo with osteoarthritis. In this disease, chondrocytes undergo a phenotypic change similar to that observed during monolayer culture, and come to ex press type I and type III collagen abundantly.

This phenomenon has been known for decades, but the exact mechanism for this phenotypic change has not been determined. In osteoarthritis, chondrocytes come to produce fibronectin abundantly while it Bosutinib clinical little exists in normal cartilage. In osteoarthritic cartilage, fibronectin therefore probably accumulates around the chondrocytes, which would activate 5B1 integrin to induce the expres sion of type I and type III collagen.

Two hours before harvesting, 100 ul of 4 uM BD Calcein AM was added to washed cells. Plates were www.selleckchem.com/products/Vandetanib.html read at 485 nm and relative fluorescence units recorded. RFU of ten replicate wells were averaged and analyzed for significance. Mann Whitney unit analysis test was applied to relative fluorescent units data from 10 replicate wells and p values are reported. mouse anti RACK1 antibody . and rabbit anti RACK1 and anti Cbp/PAG antibodies. Mouse anti Lyn, clone 10A6. 2, and MilliplexW assays were from Millipore. Horse radish Inhibitors,Modulators,Libraries peroxidase conjugated secondary anti bodies were goat anti rabbit Ig and goat anti mouse Ig antibodies, anti rabbit light chain TrueBlotWantibodies and anti rabbit light chain TrueBlot IP beadsW. Cell lysates Inhibitors or equal volumes of DMSO solvent vehicle were added to adherent, serum starved cells in 6 well plates before preparation of cell lysates.

Where indi cated, cells were stimulated with 500 or 100 ng/ml of human EGF for five ten minutes at 37 C before medium was removed, and chilled cell lysis buffer imme diately added. Dissolving cells were sonicated 15 seconds before microcentrifugation for 20 minutes. Supernatants were removed and protein concentrations quantitated Inhibitors,Modulators,Libraries using Bio Rad Bradford protein assay.Generally 20 30 ug of protein were loaded into 7. 5% Tris HCl pre cast SDS PAGE gels. MILLIPLEXW MAP 8 Plex phospho Src family kinase immunoassay Quantitative sandwich immunobead assays were used to identify Y 419 phosphorylated SFK mem bers including Src, Yes, Fyn, Fgr, Lck, Hck, Blk and Lyn.

Inhibitors,Modulators,Libraries Cell free lysates of unstimulated NSCLC Inhibitors,Modulators,Libraries cell lines were incubated with specific antibody conjugated beads which select a SFK member, followed by addition of biotiny lated pan anti phospho Src to quantify the level of Y 419 phosphorylation of that SFK Inhibitors,Modulators,Libraries inhibitor order us member. Samples were read in a luminex 100 reader after addition of PE conjugated StrepAvidin. All assays were performed and analyzed with respect to a standard curve of Hela or Ramos cell lysates according to manufacturer recom mended protocols. Western blotting SDS PAGE were performed using pre cast 7. 5% Tris HCl gel and electrophoresed in Tris Glycine SDS buffer at 100 volts for 99 minutes. Sepa rated proteins at 20 30 ug/lane were transferred to PVDF membranes using a semi dry transfer apparatus. Blotted membranes were washed, blocked overnight on a rocker at 4 C, then incubated with 1 1000 primary antibody diluted in SignalBoost, 5% BSA, or 5% milk in TBST. Secondary antibodies were added at 1 2000 for 2 hours at 25 C. ECL substrate was added, then blots exposed to film before developing.

Since CYP1A1 inducibility strongly correlates with CYP1A1 gene polymorphism fairly we also tested Inhibitors,Modulators,Libraries the genotypic asset of our cell lines regarding the two main polymorphic forms of CYP1A1. All the tested cell lines carried a wild type homozygous genotype for both the polymorphisms and so we can exclude that different genotypes are involved in the different capability of metabolizing gefitinib. The role of CYP1A1 polymorphism as a predictor of clinical outcome to EGFR TKIs in patients with advanced lung cancer has very recently been reported. The authors note that CYP1A1 2A polymorphism correlates with the response to EGFR TKIs of NSCLC, wild type T/T patients having an improved response of inhibitors versus T/C and C/C alleles.

Studies have shown that the hepatic metabolism of gefitinib is primarily catalyzed via CYP3A4, conse quently the effects of known inducers and inhibitors of CYP3A4 activity have been investigated. Our results indicate that, in NSCLC cells metabolizing gefitinib, CYP1A1 inhibition could lead to increased local exposure to the active drug. In fact, inhibition Inhibitors,Modulators,Libraries by a naphthoflavone was associated with lower gefitinib metabolism and consequently with a prolonged expo sure to locally active drug. This leads to enhanced inhi bition of EGFR, MAPK and AKT phosphorylation and cell proliferation, with the result of reduced IC50 for gefitinib in proliferation assays of EGFR wild Inhibitors,Modulators,Libraries type NSCLC cell lines. From a medicinal chemistry perspective, these results stress the importance of considering drug pharmacoki netics at the intratumoral cellular level, focusing on the roles of transport and metabolism in the target cells.

While the structure of gefitinib makes it a substrate of transporters, thus enhancing its activity toward Inhibitors,Modulators,Libraries intra cellular targets, it also harbors metabolic liabilities in tumor cells. From this point of view, its interaction with CYP3A4 seems mainly related to total body exposure gefi tinib, while CYP1A1 is mainly responsible of its metabo lism in tumor cells. A program Inhibitors,Modulators,Libraries of structural optimization should thus consider the effects of structure modulation on all these processes in combination. In addition, a strategy of increasing gefitinib activity by using specific CYP inhibitors, could be pursued in the context of optimizing the use of gefitinib for the treatment of EGFR wild type gefitinib sensitive tumors.

Interstitial lung disease has been reported as a serious adverse effect of gefitinib treatment. The incidence of acute ILD during gefitinib treatment varies between different DAPT secretase CAS ethnic groups occurring more fre quently in Japanese patients than in Caucasian. Although the precise mechanism of ILD induced by gefitinib remains unknown, it has been pro posed that bioactivation of gefitinib by CYP1A1 in the lung may be related to the risk of developing ILD mainly in smokers.

The full list from the Ingenuity pathway analysis is also included. Additionally, the IL 6 signaling pathway involving STAT3 had a significant number of contributing methylated genes, a pathway recently found to play a significant role in selleck chemicals Gemcitabine cancer stem cell regulation. Inhibitor studies further determine Inhibitors,Modulators,Libraries the role of IL 6/STAT3 pathway in invasion Based on the information generated from Ingenuity, we chose to determine how the IL 6 pathway might be regu lating this process of invasion. A number of inhibitors of downstream targets of IL 6 regulation were tested for their ability to block invasion toward SCM. We included a neutralizing antibody to interleukin 6 to test what effect this may have upstream. Downstream of the receptor, the following Inhibitors,Modulators,Libraries inhibitors were used.

the PI3K Inhibitors,Modulators,Libraries inhibitor LY294002, small molecular inhibitor of MEK called U0126 mediated responses a small molecule inhibitor of JAK called AG490 and an inhibitor of its partner signal transducers and activators of transcription 3 called Stattic. Additionally, we tested the ability of the Tec kinase family inhibitor LFM A13 based on the potential involvement of BMX during invasion. The inhibitors which demonstrated the greatest effect at blocking invasion included Stattic, LY294002, and LFM A13. However, a proliferation assay deter mined that Stattic could be preventing invasion because it was either cytotoxic to the cells or causing them to undergo apoptosis. To eliminate this possibility, viable cells were isolated after treating the DU145 cell line with Stattic for 24 hours.

These cells, although viable as deter mined Inhibitors,Modulators,Libraries by trypan blue staining, were still unable to invade. Direct interaction between the differentially methylated Inhibitors,Modulators,Libraries SOX1 and STAT3 Since inhibition of STAT3 demonstrated such a pro found effect on invasion toward SCM, we questioned its involvement with the http://www.selleckchem.com/products/Lenalidomide.html epigenetically regulated targets. Although we did not observe methylation of Stat3 itself, in both cell lines, the mRNA expression of Stat3 was increased when comparing invasive cells to their non invasive counterpart. Protein expression of pSTAT3 was also found to be increased in the invasive cells. Since both SOX1 and STAT3 are known to act as transcriptional activators after forming protein complexes with other proteins, and BMX is known to activate STAT3 itself, we determined whether STAT3 directly interacts with either SOX1 or BMX. An interaction between SOX1 and STAT3 was observed, however not between STAT3 and BMX. In addition, a significant decrease in the expression of activated pSTAT3 was seen in both sub cellular fractions of the BMX and SOX1 shRNA infected cells. However, there was no change in total expression of STAT3.

Multiple growth factors such as IGF 1, VEGF, and EGF facilitate the development and progres sion of cancer by activating PI3K pathway leading to cell survival and therapeutic resistance. Here, we showed selleck chemicals Rucaparib that Aur A was overexpressed in tongue cancer tis sue and tightly correlated with clinical stage and lymph node metastasis in patients. Thus, dys regulation of mitotic Aur A kinase and abnormal activa tion PI3K survival pathway are two essential but distinct biological processes in cancer progression. As tumorigen esis is a multiple process, combination therapeutic strate gies have shown substantially enhanced anti tumor effects and reduced side effects both in vitro and in vivo. A recent study reported that combined treatment with the pan histone deacetylase Inhibitors,Modulators,Libraries inhibitor vorinostat and Aur A kinase inhibitor MK 0457 showed a synergistic anti leukemia activity in cultured and primary AML and CML cells.

Inhibitors,Modulators,Libraries Here, we demonstrated that Aur A inhibi tory VX 680 could markedly reduce IGF 1 induced sur vival and migration. Furthermore, combinational inhibition of Aur A and PI3K Inhibitors,Modulators,Libraries showed a synergic effect in causing apoptosis and suppressing migration in cancer cells. Conclusion Taken together, our findings demonstrated that Aur A stimulated NFB signaling pathway via Akt activation to promote cancer cell survival, and formed a conceptual basis for the combination chemotherapy of targeting both Aurora kinase and growth factor induced PI3K pathway for inhibiting the enhanced survival and migration of can cer cells.

Methods Patients and clinical Inhibitors,Modulators,Libraries tissue specimens Fifty five patients who performed radical surgery were original clinically diagnosed and pathologically con firmed of TSCC between 1987 and 1992. Pertinent patient clinical reports were obtained with prior patient consent Inhibitors,Modulators,Libraries and the approval of the institutional Clinical Ethics Review Board. All of the 55 specimens and additional 30 normal adjacent tissues were collected and fixed in forma lin and embedded in paraffin in the diagnostic histopa thology laboratory at the Second Affiliated Hospital of Sun Yat sen University. Patient clinic pathological fea tures were shown in Table 1. Tumors were staged accord ing to UICC classification stage I, II, III and IV or histopathology classification stage I stage II and stage III.

Reagents and cell lines VX 680 was purchased from Kava Technology, San Diego, CA, API 2 was from Calbiochem, IGF 1 from Biosource, tumor necrosis factor and selleckchem wortmannin from Cell Signaling. Human tongue squamous cancer cell line Tca8113 was kindly provided by Xiao feng Zhu, human oral floor cancer cell line KB was obtained from ATCC. Immunohistochemical staining of Aur A expression Aur A immunohistostaining using an anti Aur A antibody on tongue cancer tissues was performed as pre viously described. Moderate or strong cytoplasm stain ing, considered as positive reaction, was assessed semi quantitatively by at least two independent pathologists.

Changes selleck kinase inhibitor in the circulat ing levels of HER receptors have been reported in a variety of different cancers. Elevated circulating HER2 levels they screening library have been identified in a group of patients with HER2 positive breast cancer with significantly Inhibitors,Modulators,Libraries worse outcome. In contrast, patients diagnosed with ovarian, head and neck, non small cell lung cancer dis play lower serum Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries HER1 concentrations, and changes of serum HER1 concentration have been correlated with the efficiency of cancer treatment. In this study, we did not observe prominent HER1 or HER2 shedding in HMEC, even in cells that overexpress HER2. Our results are consistent with a recent report that investigated HER1 shedding in malignant cells.

Their results showed Inhibitors,Modulators,Libraries that among a few malignant cell lines, HER1 shedding only occurred in cell lines that express more than 7×105 HER1 per cell and were treated for 8 h or more with phorbol 12 myristate 13 acetate. Inhibitors,Modulators,Libraries MMPs are widely expressed by many types Inhibitors,Modulators,Libraries of cells. High levels of MMPs are associated with tumor invasion and metastasis. Previously, we reported that TNF a stimulation could induce production of MMP9 in our parental HMEC, the HER1 cell line, and that this pro cess was dependent on autocrine signaling that activated HER1. In this study, we examined the secreted levels of MMP1, 2 and 9 after EGF treatment. The results show that MMP2 levels are similar among all the three HMEC lines and that EGF treatment does not increase MMP2 levels.

Thus, it appears that MMP2 secretion is not regulated by HER receptor activity.

In contrast, MMP1 expression and secretion were induced in response to HER signaling.

Notably, increased levels Inhibitors,Modulators,Libraries of the HER2 receptor enhance basal MMP1 secretion. In parental cells and HER2 cells, Inhibitors,Modulators,Libraries induction Inhibitors,Modulators,Libraries of MMP1 expression can Inhibitors,Modulators,Libraries be triggered only through ligand induced HER1 activation. Interestingly, induction Inhibitors,Modulators,Libraries of MMP9 secretion by EGF signaling was only observed in parental cells, implying the presence of HER2 or HER3 may repress MMP9 secretion in response to HER1 activation by an undefined mechanism. Our Inhibitors,Modulators,Libraries conclusion that MMP secretion is mediated by the Erk pathway is in agreement with previous reports. We also observe that the Akt pathway has a role in MMP secretion.

www.selleckchem.com/products/MLN8237.html As inhibition of MMP secretion through blockade of either pathway is only effective when secretion is stimulated by HER receptor activation, these results indicate that Erk and Akt pathways mediate MMP secretion through HER sig naling in HMEC.

Overall, these results suggest that secretion Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries thorough of these three MMP forms are regulated by distinct molecular processes and, thus, that their use as biomarkers may provide differential insight into cell sig naling processes occurring in breast cancer cells. Cytokines Inhibitors,Modulators,Libraries play an important role in the pathogenesis of cancers. Secretion of most cytokines undergo traffick ing through the Golgi, and are not dependent on proteolytic now shedding.

These slides were imaged with a Sca nArray Express HT laser scanner. ScanArray Express software was used to quantify the spot fluorescence so never intensity from the scanned Imatinib 152459-95-5 images. Spot fluorescent data were then processed and analyzed using Protein Microarray Analysis Tool and ProMAT Cali brator, both Inhibitors,Modulators,Libraries of which are open source, freeware programs that we developed specifically for processing ELISA microarray data. Standard curves were fit to a four parameter logistic model and used to estimate the individual protein concentrations in each sample. RT PCR Analysis Inhibitors,Modulators,Libraries Quantitative assessment of mRNA expression was per formed by real time RT PCR. Total RNA was extracted using RNeasy kit.

The HMECs used in the quantitative RT PCR analyses Inhibitors,Modulators,Libraries were cultured and harvested in parallel with the cells used in the com parable protein experiments in the CM and cell lysates, although the RNA studies were conducted using Inhibitors,Modulators,Libraries cells raised in seperate dishes. Inhibitors,Modulators,Libraries Complementary DNA were synthesized from total RNA via reverse transcription using ImProm II reagents and oligo Inhibitors,Modulators,Libraries dT priming. The following primers were employed in the quantitative real time Inhibitors,Modulators,Libraries RT PCR Quantification of Erk/Akt activation Phosphorylated Erk1 and Akt levels of cell lysates were quantified by conventional ELISA techniques in a 96 well microplate. R D Systems DuoSet IC ELISA kits were used in these measurements, as described before. The manufactures protocols, including the lysis buffer, were followed in all the ELISA measurements.

Before each ELISA assay, protein concentrations Inhibitors,Modulators,Libraries of the cell lysates were measured using the Bicinchoninic Acid protein quantitation kit.

Data Analysis Inhibitors,Modulators,Libraries Concentrations of secreted proteins are presented in pg per ml of conditioned Inhibitors,Modulators,Libraries medium. The Inhibitors,Modulators,Libraries total volume of medium/dish was a constant within and across Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries stu dies. Concentrations of individual proteins in cell lysates are presented in ng per mg lysate protein. We used total secreted protein in the temporal stu dies for comparisons Inhibitors,Modulators,Libraries across the cells lines. An alterna tive approach would have been to use non stimulated cells as the control, and present the difference between stimulated and non stimulated cells.

This approach was not used because the cells were first fasted overnight in serum free medium.

To continue to deprive the cells of growth factors would have led to Paclitaxel price cell death during the remaining 24 hr study, and therefore was not a reason able option.

Rather, the approach used allowed us to compare Inhibitors,Modulators,Libraries cell lines EPZ-5676 solubility in parallel and directly demonstrate the involvement of HER2 or HER3 in protein secretion following HER1 activation. Statistical differences between cell lines following EGF stimulation and/or signaling pathway inhibition were initially determined by analysis of variance, and else then delineated using Fishers protected least significant dif ference test using StatView 5. 0. 1 software. A significance level of 0. 05 was used in all cases.

Plates were then washed selleck kinase inhibitor and Inhibitors,Modulators,Libraries nuclei stained with 10 uM Hoechst 33342 in a fixation solution with 3. 7% formaldehyde. To study cell cycle arrest, HCT116 cells were incubated for 24 h with VLX40. Cells were stained using Cell Cycle Kit I reagents for DNA content and phospho histone H3 staining according to the manufacturers instructions. Primary antibodies specific for phospho histone H3, secondary antibodies DyLight 549 Conjugated Goat anti Rabbit IgG and DAPI dye were used. Processed plates were loaded in the ArrayScan and analyzed. Images were acquired for each fluorescence channel, using suitable filters with 10X or 20X objective and in each well at least 1000 cells were analyzed. Quan tification of apoptosis was performed by measuring caspase 3 activation and nuclear fragmentation, wheras quantification of cell cycle arrest was obtained by nuclear DNA content and phospho histone H3.

Flow cytometry analysis of cell cycle and apoptosis Cells were seeded in 24 well plates 24 h Inhibitors,Modulators,Libraries prior to treatment with different concentrations of VLX40 for 6, 16, 24 and 48 hours. Upon Inhibitors,Modulators,Libraries drug exposure, cells were washed with PBS and stained with Annexin Inhibitors,Modulators,Libraries V FITC according to the instructions of the vendor. Cell cycle analysis was performed by labeling digitonin permeabilized cells with 5 ug/ml propidium iodide. Flow cytometry analysis was performed using a BD LSR II flow cytometer. Phase contrast microscopy Time lapse phase contrast microscopy was performed using an automated IncuCyte phase contrast microscope. MCF 7 cells were plated on 24 well ImageLock plates and immediately placed into the IncuCyte imaging system.

The Inhibitors,Modulators,Libraries chamber is designed to fit into a standard, humidified incubator in an atmosphere of 5% CO2, and a moving objective allows the cell culture to be stationary while images are captured at different positions from well to well. Images were collected at 1 h intervals starting 30 min after placing the plate in the IncuCyte chamber and cells were left to attach for 24 h when drug treatment was performed. Cell density was calculated using the IncuCyte software. Microarray analysis RNA from cell cultures was isolated using RNeasy Mini Kit from Qiagen and immediately stored at 70 C until further use. RNA purity and quality was measured using an ND 1000 spectrophotometer and Bioanalyzer 2100, respectively. Starting from 2 ug of total RNA, gene expression analysis was performed using Genome U133 Plus 2.

0 Arrays according to the GeneChip Expression Analysis Technical Manual. Raw data was nor malized using MAS5. Connectivity Map build 02 contains genome wide expression data for 1,309 compounds. The original protocol using MCF 7 breast cancer cells as described by Lamb et al. was used. Briefly, cells were selleck chemical seeded in a 6 well plate at a density of 0. 4 106 cells per well. Cells were left to attach for 24 h, followed by exposure to either VLX40 at a final concentration of 10 uM, or to vehicle control.