Exclusive role for p55�� in BMP2 induced PI3K signalling To date, data regarding unique functions of p55�� are poor, mainly because it is speculated that the five differ Brefeldin A ent PI3K regulatory subunits have redundant functions Inhibitors,Modulators,Libraries and may compensate for each other. The data presented here show that p55�� provides specific functions during BMP2 Inhibitors,Modulators,Libraries induced PI3K signalling. This is underlined by its exclusive association with BMPRII, its BMP2 dependent phosphorylation in the iSH2 domain, and the effects on Akt phosphorylation and cell migration when knock down of p55�� was performed. We have confirmed that, besides p55��, all other class Ia regulatory subunits, namely p85 and p85B, are detectable at the mRNA level in undifferen tiated multipotent C2C12 cells.

A prom inent role for PI3K regulatory subunits during cytoskeletal rearrangements has already been described, especially in the context of actin reorganisation. Interestingly, some studies have proposed that PI3K regulatory subunits provide non redundant signalling functions dependent on their sub cellular localisation within a cell. This is in line with our data, showing Inhibitors,Modulators,Libraries that p55��, but not p85, in teracts and co localises with BMPRII, predominantly at the cell periphery. It still remains unclear how BMPRII se lectivity for p55�� over p85 is achieved. The p55�� high resolution crystal structure has not been determined and the SH2 and iSH2 domains of human p85 and p55�� share about 81. 1% sequence identity. Based on the data presented here, we now propose two possible mechanisms by which BMPRII selectivity for p55�� could occur.

First, our research revealed BMP2 dependent phosphorylation of the conserved Tyr199 within iSH2 of p55��, but not p85. Phosphorylation of p55�� iSH2 could induce struc tural changes, favouring an association of p55�� with BMPRII over that of the p85 SH2 domain. Inhibitors,Modulators,Libraries Second, the N terminal 34 residues of p55�� bind to tubulin. Be cause the p55�� N terminal sequence is unique and not present in p85, it was proposed that this interaction spe cifically recruits p55�� to the cell periphery. During onset of cortical actin rearrangements, microtubule plus ends penetrate the leading edge cytocortex together with actin nucleating factors. The binding of p55�� to mi crotubules, especially at the very tip, could thus provide a sub cellular pool of p55�� for signalling involved Inhibitors,Modulators,Libraries in cortical actin driven lamellipodia selleckchem formation. Besides specific functions of the class Ia PI3K regula tory subunits, class I catalytic subunits also attract in creasing attention to provide non redundant signalling functions. The catalytic subunit p110 has been im plicated in BMP2 induced PI3K signalling and cell mi gration by others using a pharmacological targeting approach.

It has also been studied in clinical I and II trail for its potential as an anti breast cancer drug. This query data were also applied to the original selleck chemical Lenalidomide cMap prediction, where the most up and down regulated 200 genes were used as the query signature genes. As expected, the cMap project gave a mix results in both predictions of similar effect drugs and reverse effect drugs. E2 itself only ranked 828 in the total 1309 compounds. In cMap, the rank was a summary of a drugs prediction results in every sample of all different cell lines. E2 has a lot of samples in the cMap data across all 5 cell line and the enrichment scores of these samples have large varia tions, ranging from 0. 707 to 0. 040, and this large variation led an insignificant prediction rank.

In the reverse effect prediction, Raloxi fene, anti estrogenic modulator, was found to be at rank 9 as expected, but fulvestrant, another anti estro genic modulator, only ranked 861. A closer look at the detailed cell line results revealed that fulvestrant had a negative Inhibitors,Modulators,Libraries enrichment score in the MCF7 cell line but a positive enrichment score in the HL60 cell line and the combined result led to a low rank. Over all, the comparison between prediction results of cMap and BRCA MoNet shows that BRCA MoNet adds consider able prediction power to the existent cMap data and greatly improves the prediction accuracy on both similar and reverse prediction. BRCA MoNet Application Case 2 Prediction of BMS 754807 Treated MCF7 Cell Line One additional dataset treated Inhibitors,Modulators,Libraries with drug BMS 754807 was tested against our BRCA MoNet.

This dataset came from breast xenograft MCF7 bearing mice treated with BMS 754807. MBS 784807 is a dual IGF 1RInsR inhibitor that can synergize hormonal agents and has been shown to be a potential breast cancer Inhibitors,Modulators,Libraries drug. Study Inhibitors,Modulators,Libraries showed that there is an elevated IGF IR activity specific in triple negative breast cancer and because of that, BMS 784807 could be a possible treatment for triple negative breast cancer. It has been investigated in several Phase I and Phase II Clinical Trials as an anti cancer drug. This dataset was tested against our BRCA MoNet for similar treatment effect predictions. The top ranked MoA was MoA 37. Interestingly, this MoA contains valproic acid, which is ranked number 1 among all the 504 BRCA MoNet Inhibitors,Modulators,Libraries drugs. Valproic acid belongs to a general class of drugs called anticonvulsants and was originally used as a non opioid pain reliever.

It has also been used to prevent migraine headaches. Recently, valproic acid has been shown to have great potential as an epigenetic drug for anti cancer activity through inhibiting cancer cell prolif eration in various types of cancer. This prediction result shows that both drugs with great selleck bio anti cancer poten tial are actually detected to have similar MoA by BRCA MoNet.

However, many of the BH3 mimetics that potently engage the Bcl 2/Bcl xL/ Bcl w sub class of the anti apoptotic Bcl 2 proteins often only weakly inhibit members of the Mcl 1/Bfl 1 sub class. An effective BH3 mimetic should neutralize both sub classes, as this is required for apoptosis Sodium orthovanadate to occur. We herein describe the biological characterization of our novel pan Bcl 2 inhibitor JY 1 106, which, based on a trisarylamide framework, reproduces the chemical nature and relative spatial projections of the key hydrophobic side chains on one face of the BH3 helix. JY 1 106 induces cancer cell death regard less of the Mcl 1 expression levels through intrinsic apoptosis pathways, and sensitizes tumor cells to che motherapeutic agents and to metabolic stress.

Further more, we demonstrate Inhibitors,Modulators,Libraries that JY 1 106 inhibits tumor growth in a lung cancer xenograft model, and, therefore, that helix mimicry based on a trisarylamide scaffold warrants further investigation towards the development of novel chemotherapeutics. Results Design Both faces of the BH3 helix contribute to the stabilization of the protein protein complex upon docking with the BH3 binding groove. In addition to the previously mentioned hydrophobic residues on one face of the Bak BH3 helix, Arg76 and Asp83 located on the other face of the helix are also important for binding. Thus, towards the development of potent, pan Bcl 2 antagonists, we wished to design amphipathic helix mimetics that would achieve more superior helix mimicry than ever reported before, as well as, potentially, better selectivity profiles against non Bcl 2 proteins.

We reasoned that this process would be accelerated by selecting and modifying a functional helix mimetic from the literature. Compounds Inhibitors,Modulators,Libraries based on an oligoamide foldamer strategy appeared excellent candidates, primarily owing to their straightforward chemical syntheses. A structure activity relationship analysis of the backbone of a previously reported oligoamide based Inhibitors,Modulators,Libraries helix mimetic designed to inhibit Bcl xL led to the discovery of the novel compound JY 1 106 with even greater affinity for Bcl xL. Although only the second most potent compound of the congeners synthesized, the aque ous solubility of JY 1 106 was, in our hands, Inhibitors,Modulators,Libraries greater than that of the most potent derivative, and so JY 1 106 was selected for further biological characterization.

Computational analyses of the binding of JY 1 106 to Bcl xL and Mcl 1 Molecular details of the interactions of JY 1 106 with Bcl xL and Mcl 1 were obtained by modeling inhibitor binding with these proteins based on the crystallographic orientations Inhibitors,Modulators,Libraries of the bound peptides, followed by MD simu lations. In addition, the SILCS selleck chem methodology was applied to quantify the energetic differences associated with binding to the two proteins and between the binding of JY 1 106 and its analog JY 1 106a to the proteins.

05 and P 0. 01. Results Expression and activation of multiple RTKs in ovarian cancer cells By phospho RTK assays, Calcitriol Sigma the expression and activation of EGFR, ERBB2, ERBB4 and MET were activated in SKOV3 cells, and EGFR, MET and AXL in OVCA429 cells, and EGFR in ES2 cells under serum starved medium condition. Activation and/or expression of multiple RTK EGFR, ERBB2, ERBB4, MET, and AXL in ovarian cancer cell lines were further validated by immunoblotting with phospho specific antibodies. As shown in Figure 2A, EGFR, ERBB2, ERBB4, and MET in SKOV3, EGFR, MET, and AXL in OVCA429, and EGFR in ES2 were strongly phosphorylated. EGFR, MET, and AXL activation in the ovarian cancer lines was compar able to that in MESO924 cells, which are known to feature strong activation of these RTK.

By contrast, activation of EGFR, ERBB2, MET, and AXL was weak to undetectable in Hela cells. Co activation and co expression of multiple RTKs were further con firmed Inhibitors,Modulators,Libraries in these cells by immunoprecipitation with RTK specific antibodies and immunoblotted with Inhibitors,Modulators,Libraries phosphotyr osine antibody. Immunoblotting showed strong and moderate p53 expression in ES2 and OVCA429, respectively, whereas p53 was undetectable in SKOV3. We further evaluated the simultaneous expression/ activation of multiple RTKs by immunoblotting and immunoprecipitation in 15 primary ovarian tumors including 3 non epithelial ovarian tumor, and 12 epithelial ovarian tumors. Receptor EGFR, ERBB2, MET, and AXL were strongly co activated in most primary ovarian tumors. We next compared the inhibitionary effect of tumor cell proliferation between HSP90 inhibitor 17 AAG and various individual kinase inhibitors.

EGFR, MET, and AXL signaling pathways in OVCA429 cells were blocked individually by EGFR inhibitor gefitinib, MET inhibitor PHA665752, or shRNA specific to AXL. various combi nation of kinase inhibitors were also performed, As shown in Figure 3A, the most striking reduction in cell viability was seen Inhibitors,Modulators,Libraries in cells treated Inhibitors,Modulators,Libraries with 17 AAG Inhibitors,Modulators,Libraries or com bination of all 3 kinase inhibitors with 75% cell decrease observed. EGFR and MET inhibitors alone or together had mild or little effects on cell viability. AXL inhibition by lentiviral shRNA1 and shRNA2 resulted in 50% and 25% inhibition of cell viability in OVCA429, respectively, whereas combination of EGFR/MET and AXL inhibition resulted in 65% reduction in viability. The AXL shRNA mediated knockdown resulted in 95% and 60% decrease of AXL protein expres sionin OVCA429. Inactivation of multi RTKs and downstream intermediates by HSP90 inhibition The selleck inhibitor observation that individual RTK inhibitors have little effect on cell viability, suggested that activation of any one RTK is insufficient to sustain ovar ian cancer growth and/or survival.

Clinical trials for HDAC inhibitors in breast cancer treatment, such as vorinostat, are still in early phases and often involve patients with advanced nilotinib mechanism of action disease. These studies have seen only partial efficacy that often increases when in combination with other agents, such as chemotherapy, and there are often issues of toxicity. Our data suggest that developing therapeutic agents to target the scaffolding component of HDAC complexes, such as Sin3A, may be of value, particularly because Sin3A affected only a subset of genes, but its loss still caused cell death. An agent that could disrupt Sin3A would target both its HDAC dependent and independent activities, possibly expand ing the efficacy beyond that of HDAC inhibitors. Other reports support the finding that Sin3A has both HDAC1/2 dependent and independent capabilities.

For example, in stem cells, Sin3A is the key member of the Sin3 complex Inhibitors,Modulators,Libraries involved in the regulation of NANOG gene expression, not HDAC1/2. Several studies have also shown that Sin3A can interact with histone methylases, DNA methy lation proteins, chromatin remodeling enzymes, and O linked N acetylglucosamine transferase, demonstrating that Sin3A has the potential to serve as an integrator of broad transcriptional and epigenetic Inhibitors,Modulators,Libraries changes in cells. Furthermore, in vitro transcription reactions on reconstituted nucleosomal templates find that addi tion of the HDAC inhibitor, trichostatin A, abolishes Sin3A mediated repression of an acetylated histone H3 template, but not acetylated histone H4 tem plate.

This Inhibitors,Modulators,Libraries in vitro experiment shows that Sin3A, even in the absence of other repressor molecules Inhibitors,Modulators,Libraries and enzymatic proteins, possesses some intrinsic HDAC1/2 independent capabilities. Our data show that loss of Sin3A increases apoptosis of ERa positive MCF7 cells. Upon further mechanistic experiments, we find that several genes with known roles in apoptosis are increased with Sin3A knockdown. This suggests that Sin3A normally represses Inhibitors,Modulators,Libraries their expression in MCF7 cells to aide in preventing apoptosis, and subsequently, promote cell growth. The apoptotic gene targets we identified fall into both the extrinsic death receptor and intrinsic mitochondrial apoptotic signaling pathways. Interestingly, www.selleckchem.com/products/Bortezomib.html we find that Sin3A regulates genes involved in all steps of the extrinsic pathway in MCF7 cells ligands, death recep tors, adaptors, and caspases. Specifically, levels of the TRAIL ligand, and its receptor, TRAILR1, are increased in MCF7 cells with Sin3A siRNA. TRAIL is a member of the tumor necrosis factor superfamily of cytokines which can induce apoptosis by binding to extracellular domains of one of its receptors, which includes TRAILR1, a member of the TNF receptor superfamily.

Thus GILZ overexpression induced an increase in p AKT and an enhancement of AKT activity. selleck compound AKT binding pro teins may cause structural changes and phosphorylations that activate AKT. We also revealed Inhibitors,Modulators,Libraries the presence of GILZ AKT complexes in BG 1 cells using immunoprecipi tation experiments. GILZ silencing reduces cell proliferation and AKT phosphorylation in BG 1 cells We studied the effects of knocking down GILZ mRNAs in BG 1 cells on cell proliferation and AKT activation. Real time PCR and western blot analyses revealed that siRNA duplexes efficiently and specifically inhibited the expres sion of GILZ more than 75% lower than in cells treated with irrelevant control siRNA. Silencing GILZ gene expression led to a marked inhibition of cell proliferation and AKT phosphorylation, without changing phospho ERK1/2 status.

Down regulation of GILZ expression in OVCAR3 cells, an ovarian cancer cell line that contains high amount of GILZ, also resulted in a decrease of cell proliferation. These various findings reveal a previ ously unappreciated role of GILZ in the Inhibitors,Modulators,Libraries regulation of pro liferation and AKT activation. GILZ controls p21 and cyclin D1 expression The cyclin dependent kinase inhibitor p21 and cyclin D1 are two AKT targeted proteins that negatively and positively control cell cycle progression and proliferation. Cyclin D1 activates cyclin dependent kinases, leading to phosphorylation of retino blastoma with the resulting promotion of cell cycle progression. We found that the overexpression of GILZ caused the up regulation of cyclin D1 and increased the amount of phosphorylated Rb .

in contrast, p21 was down regulated. At the opposite, down regulation of GILZ resulted in decreased amount of cyclin D1 gene products and p Rb whereas those of p21 increased. Thus the effects of down regulation of GILZ mirrored those of overexpression. GILZ caused changes in p21 and cyclin D1 expression in such a way that increases in GILZ expression would accel erate cell cycle progression. Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries To confirm this prediction we analyzed the cell cycle distribution of synchronized cells following removal of the thymidine block. We found that pGILZ cells entered S phase earlier than CTRL cells. AKT activation contributes to BG 1 cell proliferation To investigate whether AKT activation is required Inhibitors,Modulators,Libraries for con trol of BG 1 cell proliferation, we used Triciribine, a spe cific pharmacological inhibitor of AKT phosphorylation.

Triciribine treatment reduced p AKT levels and in parallel decreased spontaneous proliferation of pGILZ and CTRL clones. selleck These findings indicate that AKT phosphorylation contributes to BG 1 cell prolifera tion. Further, Triciribine also caused an up regulation of p21 expression in both CTRL and pGILZ clones. Interest ingly, cyclin D1 expression remained unchanged. In addi tion, GILZ levels remained unchanged suggesting that p AKT inhibition did not significantly affect GILZ expres sion.

Western blots were scanned and Crizotinib 877399-52-5 aligned with the Photoshop selleck 6. 0 channel mixer. Antibodies for western blots Hdac1 rabbit polyclonal 65 kDA, 1 500, Apoptosis detection and cell cycle analysis Effects on apoptosis Inhibitors,Modulators,Libraries induction were analyzed in A204 cells. Cells were incubated in 75 cm2 tissue flasks with the drugs for 24, 48 and 72 hr. A204 cells were treated with for ethanol, with SAHA, fenretinide or a combination of SAHA and fenretinide. All experiments were at least performed in biological trip licates. An annexin V FITC apoptosis detection kit was employed. Cells were washed with PBS and fluorescein Inhibitors,Modulators,Libraries isothiocyanate conjugated annexin V and propidiumiodide were added. Cells were then incubated at room temperature and analyzed by flowcytometry, using a Facscalibur.

For cell cycle analysis cells were Inhibitors,Modulators,Libraries cultured and treated with compounds as described before, incubated with DAPI and measured using the Inhibitors,Modulators,Libraries Facscalibur. cDNA microarray experiments and statistical analysis A204 cells were Inhibitors,Modulators,Libraries treated with 10 umol SAHA or equal amounts of ethanol. SAHA treated Inhibitors,Modulators,Libraries A204 cells and control samples were used as biological triplicates. After 12 h incubation cells were Inhibitors,Modulators,Libraries harvested and RNA was isolated by using an RNAeasy mini kit. Affymetrix Gene Chip human 1. 0 was used. Microarray data were analyzed using GeneSpring GX Software. Microarray data complywiththe MIAME standard. Data were corrected for background noise, Inhibitors,Modulators,Libraries normalized and summarized using ExonRMA16 Algorithm.

Following quality control was performed.

To identify differentially expressed Inhibitors,Modulators,Libraries genes in SAHA treated compared to untreated A204 cells we used an unpaired t test.

For Inhibitors,Modulators,Libraries further analysis we considered genes with a students Inhibitors,Modulators,Libraries t test p value of 0. 05 and a foldchange of 2. Prior published Inhibitors,Modulators,Libraries microarray data were used Inhibitors,Modulators,Libraries as supplied, as processed lists or downloaded from GEO. Analysis of enriched GeneSets with GSEA GeneSets were Inhibitors,Modulators,Libraries downloaded from the MSig database. To process the data, in house scripts were employed. For analysis of HDAC RNA expression we compared available data from geo database of primary rhabdoid tumors to expression data from normal brain tissue. These data were MAS5. 0 normalized.

HDACs in primary rhabdoid tumor were compared to normal brain tissue from different localizations of the brain.

Microarray data were confirmed using real time qPCR. RNA was isolated as Inhibitors,Modulators,Libraries www.selleckchem.com/products/Erlotinib-Hydrochloride.html described above from G401 cell treated with SAHA for 12 h.

http://www.selleckchem.com/products/Roscovitine.html selleck kinase inhibitor RT PCR was performed using Takara RT PCR kit according to the manufacturers protocol. For Real time PCR we used Fast SYBR green. Results HDACs are highly expressed in primary rhabdoid tumors and rhabdoid tumor cell lines Aberrant expression of different HDACs has been observed in various tumors and has been linked to tumor growth progression and poor outcome. To compare the expression of HDACs in primary rhabdoid tumors and normal brain tissue we analyzed RNA expression profiles of AT/RT tissue and normal brain tissue from datasets available in the GEO database.

Afterwards, cells were harvested and stained with trypan blue. The unstained cells were coun ted in a Neubauer chamber, and the number was ex pressed as the percentage change of control group. The IC selleck chemical Oligomycin A 50, defined as the drug concentration Inhibitors,Modulators,Libraries at which cell growth was inhibited by 50%, was assessed by SPSS 16. 0 software. All experiments were repeated at least three times. Colony formation assay PaTu8988 cells treated with SAHA for 48 h were har vest, a total of 1 103 cells per well suspended in 150 uL of Mix agar with 1. 5 mL DMEM/10% FBS were plated in 30 mm plates overlying a 1% agar DMEM/10% FBS bottom layer. After 3 weeks, colonies were photo graphed at 4��. The remaining survival large colonies were manually counted. Cell cycle assay PaTu8988 cells were grown in T75 flasks and treated with indicated dosage of SAHA for 48 h.

After the treat ment, the cells were fixed with 70% ethanol overnight at 4 C, washed Inhibitors,Modulators,Libraries with PBS, re suspended in 500 uL PBS with 100 ug/mL RNase and incubated for 30 min at 37 C. After that, 2. 5 uL of PI solution was added. The DNA contents of PI stained cells were analyzed using a flow cytometry. Cell apoptosis assay PaTu8988 cell apoptosis was detected by the Annexin V Apoptosis Inhibitors,Modulators,Libraries Detection Kit according to the manufacturers protocol. Briefly, one million cells with indicated treatments were stained with FITC Annexin V and PI. Both early and late apoptotic cells were sorted by fluorescence activated cell sorting. Cell morphologic analysis A total of 4 104 PaTu8988 cells were seeded on glass cover slips in the six well plate and treated with the indicated concentration of SAHA for 48 h.

Cells were fixed and stained with Wright Giemsa stain. The slides were photographed using oil microscopy. In vitro tube formation assay or vasculogenic mimicry assay The tumor cell formation of capillary structure in vitro was tested as we previously described. Cellular immuno fluorescence staining PaTu8988 cells were seeded on glass Inhibitors,Modulators,Libraries cover slips in six well plates and treated Inhibitors,Modulators,Libraries with described dosage of SAHA for 48 h. Cells on the cover slip were then fixed with 4% paraformaldehyde for 10 min at room temperature with out permeabilization. Slides were washed three times with phosphate buffered saline, blocked with 5% bovine serum albumin for 1 h at 37 C, followed by incu bation with the primary antibody overnight at 4 C, and the secondary antibody for 1 h at room temperature.

The slides were photographed using OLYMPUS FSX 100 microscope. MTT cell viability assay The cell viability was measured by the 3 2,5 diphenyltetrazolium brom ide method, as described before. http://www.selleckchem.com/products/17-AAG(Geldanamycin).html Briefly, the PaTu8988 cells were collected and seeded in 96 well plate at a density of 2 105 cells/cm2. Different seeding densities were optimized at the beginning of the expe riments.

100 ng of total RNA were reverse transcribed into cDNA using the qScript cDNA synthesis kit. Signal transduction pathway inhibitors HT 29 colon cancer cells were seeded into a 6 well plate at 1. 5 million cells per well and incubated Wortmannin ATM overnight. The next day, the cells were treated for 5 hours with 10 uM U0126, Inhibitors,Modulators,Libraries 10 uM LY294002, or 10 uM rapamycin. Total RNA or total protein was collected from the cells for further analysis. QPCR Primers against human PDF and MAP1D were designed using Primer Express software and synthesized by Integrated DNA Technologies Steady state mRNA levels of PDF or MAP1D were determined for all cDNAs by real time PCR using PerfeCTa SYBR Green FastMix. The cycling parame Inhibitors,Modulators,Libraries ters were 95 C for 10 min followed by 40 cycles of 95 C for 30 sec and 60 C for 1 min and a dissociation program that included 95 C for 1 min, 55 C for 30 sec, and 95 C for 30 sec ramping up at 0.

2 C/sec. One distinct peak was observed for the primer sets. For the cell lines, qPCR standards were prepared using human PDF and MAP1D full length cDNA clones Inhibitors,Modulators,Libraries from Open Biosystems. The 1010 molecules/uL standard was serially diluted to 102 molecules/uL. The standards were run alongside the cDNA from the human cell lines in order to approximate the copy number of PDF or MAP1D in these cells. For the cDNA panels, fold change in mRNA expression was calculated by comparing normalized threshold cycle numbers in the cancerous tissue compared to the normal tissues. The cell experiments were performed in triplicate.

SDS PAGE and western blotting Cell pellets or human tissue samples from the VA Hospital were lysed using an SDS lysis buffer containing protease and phosphatases inhibitors. Samples were briefly sonicated to dissociate cell membranes. Fifty ug of total protein isolated from the human cell lines or tissues were separated Inhibitors,Modulators,Libraries on 10% SDS polyacrylamide gels at 100 V for 1 hr. Proteins were transferred to nitrocellulose membranes at 100 V for 75 min at 4 C. Blots were then probed overnight at 4 C with primary antibodies. The PDF antibody was a kind gift from Carmela Giglione and Thierry Meinnel. The MAP1D antibody was obtained from R D Systems. The total and phosphor ERK antibodies were purchased from Cell Signaling. The next day, blots were rinsed with 1X TBS tween and probed with anti rabbit secondary antibody for 1 hr at room temperature.

The western Inhibitors,Modulators,Libraries blots were analyzed using SuperSignal West Pico Chemiluminescent Substrate and images cause captured using the MultiImage Light Cabinet. PDF levels were normalized to B actin expression. Immunoblots were performed in triplicate. Toxicity assay Hs578Bst, Hs578T, CCD 18Co, HT 29, PrEC, and PC 3 cells were plated in 96 well microplates in growth medium at a density of 5,000 cells/well and incubated for 24 hours. The cells were then treated for 4 days with 0 250 uM actinonin.