Overe pression of PDK1 has been reported to correlate with tumor

Overe pression of PDK1 has been reported to correlate with tumor progression. We found that overe selleck chemicals Calcitriol pression of PDK1 abrogated the effect of ciglitazone on cell growth and caspase 3 7 activity. Transfection with PDK1 e pression vector was confirmed by Western blot. Together, this suggested that ciglitazone not only inhibited growth but also increased apoptosis of lung cancer cells through, at least in part, the inhibition of PDK1. The role of AMPK and SAPK JNK in mediating the effect of ciglitazone on PDK1 protein e pression Studies by this group and others also demonstrated a role for AMPK in mediating the effect of PPAR�� ligands, such as thiazolinediones compounds, in different cell systems. We showed that ciglitazone increased phosphorylation of AMPK and SAPK JNK with ma imal effect observed at 2 4 h in H1650 cells.

Interestingly, the inhibitors of AMPK, compound C, but not of SAPK JNK, SP600125, blocked the inhibitory ef fect of ciglitazone on PDK1 protein e pression in both H1650 and H1299 cells. Similarly, silencing of AMPK abrogated the effect of ciglitazone on PDK1 protein. This indicates the specificity of AMPK activation in this process. Interestingly, com bination treatment of ciglitazone and metformin, an ac tivator of AMPK, further reduced the PDK1 protein e pression. Ciglitazone decreases PDK1 promoter activity independent of PPAR�� activation We also e amined if the effects of ciglitazone on PDK1 e pression occurred at the transcriptional level. As shown in Figure 4A, the PDK1 gene promoter contains multiple transcription factor binding sites including PPRE, Egr 1, nuclear factor ��B and p53, among others.

We found that NSCLC cells transfected with wild type PDK1 promoter luciferase reporter construct showed decreased activity when e posed to ciglitazone. As e pected, Anacetrapib metformin enhanced the inhibitory effect of ciglitazone. Ne t, we assessed whether PPAR�� activation played a role in mediating the effect of ciglitazone on PDK1 pro moter activity. The effect of ciglitazone on inhibition of PDK1 promoter activity was not abrogated by PPAR�� siRNA. Note that PPAR�� siRNA blocked PPAR�� protein e pression. As e pected, we found that compound C re duced the effect of ciglitazone on PDK1 promoter activity. The role of transcription factor Egr 1 in mediating the effect of ciglitazone on e pression of PDK1 and cell growth We further tested the role of the transcription factors in mediating the effect of ciglitazone on PDK1 e pression in human lung carcinoma cells. We showed that ciglita zone significantly induced the e pression of Egr 1 protein in a time dependent manner, while it had little effect on p65 and p53. Note that a synergy was observed in the combination of ciglitazone and met formin treatment.

However, no difference in the susceptibility of RhoH cells was fo

However, no difference in the susceptibility of RhoH cells was found compared to con trol cells. Overe pression of RhoH leads to the upregulation of p21Cip1 and p27Kip1 We therefore reasoned that RhoH may play a role in regulation Axitinib cancer of cell cycle progression, rather than apopto sis. The activities of cell cycle regulating cyclin depen dent kinases are negatively controlled by the WAF CIP family of CDK inhibitors. We e amined the e pression levels of the cyclin dependent kinase inhibi tors p21Cip1 which is known to be a STAT1 induced gene and p27Kip1 of the same family by quantitative real time PCR using GAPDH as a reference gene. Total RNA was prepared from control cells and RhoH cells and the gene e pression of p21Cip1 and p27Kip1 were determined.

RhoH cells showed a 55 and 40 fold increase in the e pression of p21Cip1 and p27Kip1, respectively, compared to control cells. There were no significant changes in the e pression of p21Cip1 and p27Kip1 detectable in siRhoH cells compared to the control. This increased e pression was also found in IL3 treated bone marrow cells from wildtype compared to RhoH deficient mice. To corrobo rate this finding also on the protein level, we prepared whole cell lysates from all three cell lines and subjected them to western blot analysis using p21Cip1 and p27Kip1 specific antibodies and b actin as a loading control. Again, we found p21Cip1 and p27Kip1 to be upregulated in cells overe pressing RhoH. The result ing quantification of the blots shows a statistically signif icant two fold upregulation of p21Cip1e pression.

The detected p27Kip1 e pression is less prominent but reproducible. We therefore propose that the e pres sion of RhoH modulates IL3 induced proliferation through upregulation of p21Cip1 and p27Kip1e pression and we suggest that this is a STAT1 dependent event. Undere pression of RhoH enhances STAT5 activity It was recently shown that reduced RhoH levels can be connected to cancer and protection from apoptosis. STAT5 is the major STAT protein activated by IL3 and is described to induce anti apoptotic genes and cell proliferation. Consequently, dominant negative STAT5 leads to partial inhibition of IL3 induced prolifera tion. We therefore e amined the ability of RhoH or siRhoH cells to activate STAT5 after IL3 stimulation. Equal cell numbers of previously IL3 depleted BaF3 cells were stimulated with 50 ng ml IL3 and STAT5 was preci pitated from the resulting lysates.

Western blot analysis and quantification of pSTAT5 levels that was corrected for the level of total STAT5 showed a strong reduction of STAT5 tyro sine phosphorylation compared to control cells. This again corroborated our finding that proliferation in response to IL3 is Brefeldin_A decreased in RhoH overe pressing cells. Reduction of RhoH e pression in siRhoH cells led to a small increase in STAT5 tyrosine phosphoryla tion compared to control cells that showed higher varia tions between independent e periments.

Six basic patterns of expression could be generated on the basis

Six basic patterns of expression could be generated on the basis of the above definitions, 1 induced expression in only one treatment, 2 induced expression in two treatments and 3 induced expression in three treatments. A total of 50 different patterns of expression were produced when all four stress treatments selleck chemical analyzed in this study were accom modated into the above basic patterns. Results and Discussion Roche GS FLX and GS FLXTM sequencing and assembly Six sequencing runs yielded 910 Mb total data size equivalent to 2,913,966 raw reads. The raw sequence files are available from the NCBI Sequence Read Archive at Traces sra sra. cgi study SRP006173, as files SRR172675, SRR172676 and SRR183482, SRR172677, SRR172678 and SRR183483, SRR172679 and SRR172680.

Length frequency distribution of raw reads clustered around the 200 to 300 bp and 300 to 400 bp range as the result of using two different platforms for sequencing. A total of 2,700,168 reads entered into the assembly process which yielded 21,207 high qual ity assembled sequences. These ranged in length from 80 to 3,379 bp and had an average sequence length of 1,014 bp and 930 bp. A total of 178,636 reads remained as singletons, of these, only 5,113 clean sequences remained after quality control. Isotigs were further incor porated into 15,667 isogroups. A status summary of the sequencing, assembly and annotation process is presented in Table 1. Annotation of A. hypochondriacus contigs isotigs All contigs isotigs were queried against the nr, TAIR, UniRef100, UniRef50 and Amaranthaceae ESTs and PFAM databases for annotation.

Approximately 82% of all entries produced significant hits when queried against the nr database. The 3,901 sequences with no significant hit versus the nr database were queried against the PFAM protein domain database in order to determine their putative function. Only a small fraction of these sequences produced signifi cant hits to known protein domains. These results are available in Additional file 1. Annota tion of the 5,113 clean singletons against the TAIR data base yielded approximately 1,000 significant hits. The best hit for each unigene queried against the TAIR database was utilized to assign functional GO annotation in terms of biological process, molecular function and cellular component groups. The results are summarized in Figure 2.

As expected, the lar gest percentage in each GO group was conformed by contigs isotigs with an unknown func tional annotation. No obvious differences in the number of sequences assigned to each category, including response to biotic stress, were observed between grain amaranth and Arabidopsis thaliana. This was probably a reflection Drug_discovery of Arabidopsis known capacity respond strongly to abiotic and biotic stresses at the transcriptional level.

The glycolysis pathway and the TCA cycle were both transcriptiona

The glycolysis pathway and the TCA cycle were both transcriptionally repressed. It remains to be determined if shutting down both these pathways is part of the host response to control the repli cation of intracellular bacteria or a strategy adopted by the pathogen to survive intracellularly. Nilotinib IC50 In addition, we found that expression of 37 cytochrome P450 related genes was suppressed in the liver over the course of infection, most notably at 24 hpi. The expression of the detoxification enzymes amine UDP glucuronosyltrans ferases and N sulfotransferase was also down regulated. Our data suggests that B. pseudomallei induced impaired liver detoxifying activity might be a causative factor in liver sepsis. Collec tively, the data presented here suggests that hepatocytes, via receptors for many pro inflammatory cytokines, mod ify their metabolic pathways in response to B.

pseudomallei acute infection. Conclusion This genome wide expression profile demonstrates that a general alarm signal of infection is triggered by the host upon infection with B. pseudomallei and subse quently various defence programs are activated to con trol the replication of the intracellular pathogen. Nevertheless, the overwhelmed inflammatory response to infection as well as tissue injury leads to metabolic disturbances and homeostatic imbalance which is detri mental to the host. The suboptimal complement func tion correlates with uncontrolled spread of the bacteria, a hallmark of the acute nature of this infection. In addi tion, we postulate tissue damage following B.

pseudo mallei acute infection is contributing to dysregulation of the innate immune response via TLR2, the surveillance receptor that recognizes both endogenous and exogen ous molecules. Animals 7 to 9 week old BALB c mice were purchased from the Institute for Medical Research, Malaysia. They were housed in High Temperature Polysufone cages with a bedding of wood shavings, subjected to a 12 hr light dark cycle and fed on a diet of commer cial pellets and distilled water ad libitum. All animal experiments were performed in accordance with the Universiti Kebangsaan Malaysia animal ethics guidelines and approved by the Universiti Kebangsaan Malaysia Animal Ethics Committee. Bacteria The three clinical B. pseudomallei isolates used in this study are listed in Table 2. All B.

pseudomallei isolates were pre viously characterized based on biochemical tests as well as by 16 S rRNA sequencing. Genome comparison with B. pseudomallei strain K96243 and B. thailandensis strain E264 identified B. pseudomallei D286, R15 and H10 as members of the YLF genomic group. Bacteria were grown in Brain Heart Infusion broth overnight at Carfilzomib 37 C. The cells were centrifuged at 10,000 �� g, suspended in BHI broth con taining 20% glycerol, frozen immediately in aliquots of 109 CFU per ml and stored at 80 C.

For example, the synthesis of a ketoglutarate through transaminat

For example, the synthesis of a ketoglutarate through transamination reactions could be used in the TCA cycle to provide energy. Interest ingly, we found over expression of genes selleck chemicals Regorafenib encoding enzymes involved in the TCA cycle, such as succinate dehydrogenase, isocitrate dehydrogenase and malate dehydrogenase, in fish fed VD. This stimulation of the TCA cycle could be related to the higher levels of ATP required for LC PUFA and choles terol biosynthesis in fish fed VD. Since marine fish have a low capacity to digest complex carbohydrates, in con trast to mammals, the use of proteins as an essential source of energy can thus explain the stimula tion of the amino acid metabolism in fish fed VD. As shown in rainbow trout fed on a vegetable based diet, the lower growth rate in fish fed VD in the present study could be associated with higher proteolytic activity compared with fish fed FD.

Interestingly, while both half sibfamilies G and g exhibited similar proteolysis regulation, the expression of several genes involved in macromolecule biosynthesis, and particularly in protein biosynthesis, were up regulated in half sibfamily G. This result, suggesting a higher protein turnover in half sibfamily G compared with half sibfamily g when fish were fed VD, could be related to the higher growth rate observed in half sib family G fed VD. As protein biosynthesis requires energy from ATP hydrolysis, the higher protein bio synthesis in half sibfamily could be related to a higher activity of mitochondrial ATP production.

Accordingly, genes involved in ATP biosynthesis and ATP synthesis coupled electron transport were found up regulated in half sibfamily G. The diet �� half sibfamily interaction that we found for the expression of genes involved in aromatic amino acid metabolism rein forces the difference in protein metabolism between the two half sibfamilies. In the present study, the vegetable diet used, based on linseed oil, was characterised by a very low ARA content and poor levels of n 3 LC PUFA. Eicosanoids derived from ARA are known to be involved in the proliferation of hepatocytes and immune cells. As a conse quence, the lower hepatosomatic index measured in fish fed VD could be linked to a lower hepatocyte proliferation due to a deficiency in ARA, which was sup ported by down expression of 32 genes involved in cell proliferation in this dietary group.

Moreover, fatty acid imbalances can induce an immune deficiency in all ver tebrates including fish. In particular, the �� n 3 �� n 6 fatty acid ratio is considered as a key element regulating immune cell structure, Cilengitide cell signalling, and eicosanoid production. The present microarray data revealed genes of the immune system, particularly the innate immune response, exhibiting lower expression in fish fed with VD.

The cells were then fixed with 4% paraformalde hyde for 30 min at

The cells were then fixed with 4% paraformalde hyde for 30 min at room temperature, treated with 2% H2O2 for 30 min followed by 0. 3% Triton X 100 for 30 min, and then stained with the following antibodies anti Oct3/4, anti CD133, anti BCRP, and anti ALDH. The negative control omitted the primary anti body. Ku-0059436 Cells were incubated in a humidified box at 4 C overnight, and then the secondary antibody was added to the cells, followed by incubation at room temperature for 30 min. After the reaction with DAB, the cells were smeared onto slides, examined by microscopy and photographed with a digital camera connected to the microscope. Cell cycle and apoptosis analyses Cell cycle and apoptosis analyses were both performed after 24 hours of sorting.

The cell cycle was examined using a CycleTEST PLUS DNA Reagent Kit follow ing the manufacturers instructions. Cell apoptosis was examined using an Annexin V FITC Apoptosis Detection Kit following the manufac turers instructions. Briefly, the cells were counted and 5 105 1 106 cells of each group were centrifuged at 179 g for 10 min. After the supernatant was removed, cold PBS was added to the cell pellet, followed by gentle vor texing to resuspend the cells. Then, the cells were washed twice, resuspended in 200 uL binding buffer containing 10 uL annexin V FITC, and gently mixed and incubated at room temperature for 15 min while protected from light. Finally, 300 uL binding buffer and 50 uL PI were added to the cell suspension, followed by flow cytometric analysis. Each sample was prepared in triplicate.

In vivo xenografting in immunodeficient mice NOD/SCID mice were purchased from the Animal Insti tute of the Chinese Academy of Medical Science and Peking Union Medical College 2009 0004 and maintained in microisola tor cages. All experiments were approved by the Animal Care Committee of CAMS and PUMC. Freshly sorted SP and non SP cells in 200 ul Matrigel diluted in PBS at a 1 1 ratio were injected subcuta neously into the left axillary fossa of female NOD/SCID mice. Groups of mice were inoculated with SP or non SP cells at 1 105, 1 104 and 2 103 cells, respectively. Tumor appearance was inspected weekly by visual observation and palpation. Mice were sacrificed after 8 weeks and the tumors were harvested, measured, and photographed.

Tumor volumes were mea sured using a digital caliper and approximated according to the formula V 1 2ab2, where a and b are the long and short diameters of the tumor, respectively. Tu mors were fixed in 10% buffered formalin, embedded in paraffin, and then sections were prepared for H E staining. Chemoresistance analysis The sensitivities to chemotherapeutic reagents of HeLa, SP, and non SP cells were assessed using an MTS assay. Briefly, 2 103 cells per well were seeded on 96 well plates in 200 ul per well of appropriate growth Dacomitinib medium. After 24 hours, the cells were treated with TSA at vari ous concentrations.

and expression of cleaved caspase 3

and expression of cleaved caspase 3 Ceritinib supplier and cleaved PARP were measured by immunoblot. Results showed that apoptosis of cells increased, viability of cells reduced, levels of cleaved caspase 3 and cleaved PARP increased significantly after treated with palmitate. However, when pretreated with adiponectin, we found that adiponectin pretreatment sig nificantly decreased apoptosis of cells, increased viability of cells, reduced the level of cleaved caspase 3 as well as cleaved PARP. These results indicated that adiponectin might attenuate palmitate induced apoptosis in H9c2 cells through reducing the activation of caspase 3 and PARP. PI3K/Akt was involved in the process of adiponectin mediated anti apoptosis Adiponectin is also known to activate PI3K/Akt signaling pathway, and the involvement of this signaling pathway in suppressive effects of adiponectin on palmitate induced apoptosis was investigated by PI3K inhibitor, LY294002.

The level of p Akt was decreased after exposure of H9c2 cells to palmitate for 12 h. Simultaneously the level of cleaved caspase 3 and cleaved PARP was increased significantly. Cells were first pretreated with 2. 5 ug/mL globular adiponectin, then treated with palmitate for 12 h, and lastly assayed by immunoblot. Results showed that the level of p Akt decreased dra matically after treated with palmitate. However, its level restored to the control level after pretreated with 2. 5 ug/mL globular adiponectin. To test whether PI3K/Akt signaling pathway was involved in the inhibitory effect of adiponectin on palmitate induced apoptosis in H9c2 cells, we used the inhibitor of PI3K/Akt, 10 uM LY294002 reference from.

Cells were first pretreated with 10 uM LY294002 for 1 h, then treated with 2. 5 ug/mL globular adiponectin for another 1 h, and lastly treated with palmitate for 12 h. Results showed that the restored level of p Akt induced by 2. 5 ug/mL globular adiponectin was decreased again, and levels of cleaved caspase 3 and cleaved PARP were also reversed after pretreated with LY294002 compared with 2. 5 ug/mL globular adiponectin plus palmitate group. Taken to gether, these results demonstrated that adiponectin partially inhibited palmitate induced apoptosis in H9c2 cells via activat ing the PI3K/Akt signaling pathway.

ERK1/2 was also involved in the process of adiponectin mediated Batimastat anti apoptosis In the present study, results showed that the level of p ERK1/2 increased significantly when treated with palmitate for 12 h whereas the level of p ERK1/ 2 decreased significantly and almost restored to the normal by pre incubation with 2. 5 ug/mL globular adiponectin. Taken together, these results suggested that adiponectin sup pressed palmitate induced apoptosis through reducing the activity of ERK1/2 signaling pathway. In order to further determine the role of the ERK1/2 in palmitate induced H9c2 cells apoptosis, we used its inhibitor, 10 uM U0126 reference from.

In in vitro treatment, whole cell extracts from intact cells were

In in vitro treatment, whole cell extracts from intact cells were treat ed with wortmannin during heating. Cell survival assay Cell survival selleck chemicals Vandetanib after heating at 44?C for 0, 15, 30, 60, 90 or 120 min was quantitated by plating cells into 25 cm2 flask containing the medium. Ten to fourteen days later, cell colonies were rinsed with PBS, fixed with methanol, stained with 2% Giemsa solution. Colonies containing at least 50 cells were count ed. The number of cells per colony was determined prior to experiment. Western blotting analysis Detailed procedure of Western blotting is described else where. Aliquots of whole cell extracts were used for Western blotting analysis of Bax and p53. After electrophoresis on 15% polyacryla mide gels containing 0.

1% SDS and electrophoretic transfer onto Poly Screen PVDF membranes, the proteins on each membrane were incubated with the anti human Bax polyclonal antibody Ab 1, anti human p53 monoclonal antibody DO 1, anti human phosphorylated p53 polyclonal antibody Phospho p53 or anti human WAF1 monoclonal antibody EA10. The bands were visualized using horseradish peroxidase conjugated goat anti rabbit or anti mouse IgG anti body and the BLAST Blotting Amplification System. Preparation of nuclear or whole cell extracts for gel mobil ity shift assay Nuclear extracts were prepared from A 172 transformed cells 6 hr after heat treatment, heat and glycerol treat ments or no treatments as in vivo treatment samples. As in vitro treatment samples, whole cell extracts were prepared from intact A 172 transformed cells suspended in extrac tion buffer, 0.

5 mM phenylmethyl sulfanylfluo ride, 25% glycerol, 1. 2M spermidine and were treated with glycerol, heat or combination of glycerol and heat, and subsequently incu bated for 30 min at 37?C. The procedures of nuclear pro tein extraction are described previously. Shortly, the cells were washed with PBS and suspended in washing buffer and then homogenized on ice in hy potonic buffer, and 0. 5 mM phenylmethyl sulfanylfluoride with a hand driv en Dounce homogenizer. The homogenates were centri fuged to precipitate the nuclei, which were resuspended in extraction buffer glycerol, 1. 2M sper midine. The resulting nuclear suspensions were centri fuged to precipitate the chromatin and the nuclear extracts were collected and dialyzed against binding buffer glyc erol. The protein concentration of each extract was quan tified using a BIO RAD Protein Assay Kit with bovine serum albumin as the standard. Gel mobility shift assay The p53 p53CON binding activity was measured by a gel shift assay using a synthetic double stranded DNA frag ment encoding the p53CON on the Drug_discovery upstream of bax gene as a probe. Detailed procedure is described elsewhere.

We confirmed association of endogenous b arrestins with AMPK and

We confirmed association of endogenous b arrestins with AMPK and CAMKKb in fat explants, where we immu noprecipitated AMPKa1 and probed western blots with b arrestin 1 2 or CAMKKb antibodies. AMPK could be co immunoprecipitated with CAMKKb and b arrestin 2. Therefore, we conclude that b arrestin 2 www.selleckchem.com/products/Axitinib.html might form an inhibitory complex with AMPK and its upstream kinase, CAMKKb. b arrestin 2 directly inhibits CAMKKb activity in vitro To examine whether b arrestin 2 can directly inhibit CAMKKb activity, thus preventing phosphorylation of AMPK, we incubated recombinant GST tagged b arrestin 2 or GST alone with recombinant CAMKKb in the presence of 32P ATP and the substrate myelin basic protein. CAMKKb activity was determined by quantifying incorporation of 32 P into MBP.

Reactions were performed with 50ng CAMKKb and carried out for 15 minutes, which resulted in maximal MBP phosphorylation. Phosphorylation of MBP by CAMKKb was inhibited in a dose dependent fashion upon addition of b arrestin 2 GST but not GST alone, sug gesting an overall inhibitory effect of b arrestin 2 on CAMKKb activity. We then specifically examined phos phorylation of AMPK on Thr172. CAMKKb was incu bated with recombinant heterotrimeric AMPK in the presence and absence of 500pM GST b arrestin 2 or with GST alone, and phosphorylation determined by western blot using anti phospho AMPK and anti total AMPK. CAMKKb stimulated AMPK phosphorylation was abolished by addition of recombinant GST b arrestin 2, but not GST. Discussion Here we describe a novel role for b arrestin 2 in the regulation of AMPK, downstream of PAR2.

We demon strate that PAR2 can activate AMPK in the presence of low b arrestin 2 levels, and inhibit it in cells with high levels of b arrestin 2. While previous studies have inves tigated the mechanism of AMPK activation by another proteinase activated receptor, PAR1, those studies did not deal with b arrestins. Furthermore, the role of b arrestins in signaling by the two receptors is quite different. PAR2 activation of AMPK involves the Ca2 sensitive enzyme, CAMKKb, while the inhibitory path way involves b arrestin dependent suppression of this same activity. As was observed for PAR1, LKB 1 may also play a role in PAR2 stimulated AMPK activation, but the sensitivity of this enzyme to b arrestin dependent regulation remains to be investi gated.

Research by ours and other Carfilzomib groups over the last few years has revealed that b arrestins can direct signals that oppose, facilitate, or act independently of a number of G protein directed signals. With respect to PAR2, we have shown that Ca2 mobilization, down stream of Gaq activation, promotes nuclear MAPK activity, PI3K activity and LIMK activation, while b arrestins promote inhibition of PI3K and LIMK and membrane sequestration of MAPK activity.

We should note that the

We should note that the Navitoclax Phase 2 equilibrium state of the network 1100 has 0 for the tumor state. This is because the tumor is activated by K3 and inhibition of K3 should eradicate the tumor. On the other hand, since both K1 and K2 can cause tumor through activation of intermediate K3, inhibition of only one of K1 and K2 will not block the tumor. The BN following inhibition of K2 is shown in Figure 7 where the attractor 1011 denotes a tumorous phenotype. Experiment design to infer the dynamic pathway structure The TIM can be used to produce possible dynamic models based on assumptions of latent activa tions or mutations. For instance, knowledge of the steady state value of the target K1 following application of target inhibitor for K3, will remove one of the possibilities.

Fol lowing inhibition of K3, the value of K1 will remain 1 for the case of Figure 4 as K1 is upstream of K3. Conversely, the value of K1 will be 0 for the second case as K3 activates K1. In the following paragraphs, we will consider a gen eral pathway obtained from a TIM having the structure shown in Figure 8 but with unknown directionalities of the blocks and target positions. For the current analy sis, we will assume that there are no common targets 1011 be located down stream of Bi. Note that the number of experiments required is based on steady state measurements following particular per turbations. Time series measurements can reduce the 0100 01 number of experiments required but may not be always technically feasible.

For our analysis, we are assuming that we can inhibit specific targets of our choice and we can measure the steady state target expression following applicatin of the target inhibitions. We can locate the directionality of the blocks B1 to BL by using at most L ? 1 steady state measurements. We can start by randomly picking any block Bi and blocking the targets in that block, the blocks that will remain acti vated will be upstream of that block and the blocks that The next step will be locating the directionality of tar gets in each parallel line of the block. We can start with an experiment where for each block Bi, one target from each line up to a maximum of ai ? 1 lines will be inhib ited. We Cilengitide cannot inhibit all the lines in a block or else the downstream blocks will also be inhibited and no infer ence can be made on those blocks for that experiment. While locating the directionality of the serial blocks Bi, we have already validated the position of one target from each parallel line in a serial block.