For the F4ac ETEC infection, the responses of the host cells were characterized by great up regulations on immune, wound ing and inflammatory response. The findings herein pro vided a solid proof why ETEC with F4 may be more virulent compared to F18 which seems to elicit necessary milder effects, which further characterized and defined the gen etic mechanisms of responses to different ETEC colonization and adhesion in small intestine of piglets. Materials and Methods Cell culture The IPEC J2 cell line was grown in Dulbeccos modified eagle medium Hams F 12 medium supplemented with 5% fetal calf serum and was maintained in a 95% air 5% CO2 humidified atmosphere at 37 C, which were free of mycoplasma contamination. Bacterial strains F4ab ETEC strain 195 and F4ac ETEC strain 200 were removed from cryo storage and cultured in Ordin ary Broth Agar at 37 C for three generations.

ETEC strain 8813 was cultured in static Tryp tone Soya Agar medium at 37 C for 24 h, and then in static Tryptone Soya Broth medium at 37 C for two generations. For cell infection experiment, the E. coli strains were subcultured in shaking LB and TSB medium, respectively, at 37 C for 12 h, then centrifuged and washed with sterile PBS. Finally the bacterial suspension was prepared in PBS. Infection of the cell lines Monolayers of cells prepared in 24 well tissue culture plates were washed twice with PBS, then 0. 5 ml of DMEM was added. A total of 20ul of bacterial suspension was used for infection or the same volume of PBS as control. The cells were incubated at 37 C in a 95% air 5% CO2 air atmosphere for 3 h.

The adhesion values of the ETEC strains to IPEC J2 cells were checked by real time PCR with slightly modified procedures described by Candela et al. Twelve samples were prepared including nine with the three ETEC strains infection treatments and three samples as control. Total RNA isolation IPEC J2 cells infected with and without E. coli strains were washed twice with PBS, then lysed with TRIZOL Reagent directly in the culture dishes. Isolation of RNA was performed using TRIZOL Reagent following the manufacturers instructions and checked for a RIN number to inspect the RNA integration by an Agilent Bioanalyzer 2100. Qualified total RNA was further purified by RNeasy micro kit and RNase Free DNase Set.

Sample labeling and hybridization Total RNA was amplified and labelled by Low Input Quick Amp Labeling Kit, One Color, following the manu facturers instructions. The labeled cRNA was purified by RNeasy mini kit, then used for hybridization onto porcine oligo microarray slides containing 43,603 oligonucleotide probes at 65 C for 17 h. The hybri Batimastat dized microarray slides were washed according to the manufacturers instructions and were scanned by Agilent Microarray Scanner at 5 mm resolution. Raw data were normalized by Quantile algorithm, Gene Spring Soft ware 11. 0.

Notably, we found that the common G allele of the IL 6 pro moter variant showed association with poor sur vival of patients with advanced gastric cancer treated with palliative chemotherapy. The minor IL 6R C allele showed a weaker selleck chemicals prognostic role than the IL 6 promoter variant. However, in support of a dynamic modulation of the IL 6/sIL 6R system, we observed a possible additive ef fect with worst survival outcomes in the presence of both IL 6 and IL 6R unfavorable genotypes. The different distribution of patients with and without liver metastasis according to the sIL 6R genotypes would also support the role of the IL 6/IL 6R system in the ac quisition of a specific pattern of metastatic spread. In experimental and in vivo models, IL 6 increases the metastatic potential of circulating tumor cells and mod ulates tissue homeostasis in a target organ of metastasis such as the liver.

Also, sIL 6R mediated trans signaling displays pro invasive and pro metastatic signals. It is maximized in rs8192284 IL 6R minor allele carriers and it is likely to promote hematogenous spread, causing a specific pattern of metastatic disease. The common G allele of the rs1800795 IL 6 promoter variant showed association with unfavorable survival out comes of patients with ovarian cancer, breast cancer, neuroblastoma and hematologic malignancies. To the best of our knowledge, there is only one pub lished study reporting the results of a prognostic analysis of IL 6 polymorphisms in gastric cancer patients.

Liao et al showed a significant association between high IL 6 circulating levels and poor survival of stage II III, sur gically resected patients, but the rs1800796 IL 6 variant did not show prognostic role. Notably, they could not in vestigate the IL 6 rs1800795 because of the rarity of the variant allele in Asiatic populations, while the func tional effects of the IL 6 rs1800796 are less extensively studied compared with the IL 6 rs1800795. Less information is available on the clinical impact of the rs8192284 IL 6R genetic variant. In multiple myeloma patients the minor rs8192284 C allele showed association with lower overall survival, but in neuroblastoma Table 2 Results of the multivariate cox proportional to explain different sensitivity and clinical outcomes of patients treated with novel target therapies.

At the same time, IL 6/IL 6R analyses could offer the opportunity of developing an alternative therapeutic strategy. In pa tients with metastatic renal cell cancer, high IL 6 serum levels were predictive of improved progression free sur vival from the multi kinase inhibitor Pazopanib com pared with placebo. In experimental models, IL 6 showed induction of cancer stem cells and epithelial mesenchimal transition phenotype, which are possible condition for resistance to the anti HER 2 compounds trastuzumab and lapatinib. High IL 6 levels showed association with toxicity from Vorinostat in prostate cancer GSK-3 patients.

Total protein was extracted from cultured cells by adding 2X sample buffer, 20 mM Tris HCl pH 7. 4, 5 mM mag nesium chloride, 10 ug/ml complete protease Rapamycin clinical trial inhibitor cocktail, 1 mM phenylmethylsulfonylfluoride. DNA was shared by pipetting up and down for 3 minutes at room temperature. Samples were boiled at 95 C for 15 minutes, centrifuged at 13,000 rpm for 10 seconds and then sub jected to 14% SDS PAGE. After blocking overnight at 4 C in a buffer containing PBS, 0. 1% Tween 20 and 5% low fat milk powder, nitro cellulose membranes were incubated for 90 minutes with primary antibodies. Antibodies against DNMT1, DNMT3a, DNMT3b, APC, RASSF1A and B actin were used. Membranes were washed three times for 10 minutes in a buffer containing PBS and 0.

1% Tween 20 and were incubated with a peroxidase coupled secondary antibody to visualize responsive bands after incubation with West Pico lumi nescence substrate. Densitometry analysis was performed by peak intensity analysis on a GeneGnome image capture and analysis system. Bands were normalized to B actin expression which was used as an internal loading control. Immunohistochemistry Formalin fixed and paraffin embedded xenograft tumour samples were cut into 5 um sections deparaffinised using graded alcohols. Antigen retrieval was performed by heat induced epitope retrieval in pH 9 antigen retrieval buffer at 95 C for 60 minutes. Endogenous peroxidase blocking was carried out for 10 minutes with peroxidase blocking reagent. Subsequently, the primary antibody against DNMT1 and DNMT3a was applied for 30 minutes at RT.

For detection of the primary anti bodies the ready to use REAL EnVision Detection System was used in accordance with the manu facturers instructions. The EnVision staining system is based on an HRP labeled dextran polymer, which is con jugated to secondary antibodies eliminating the nonspe cific staining background resulting from endogenous avidin biotin activity. Visualization was performed using diaminobenzidine as the chromogen substrate being a part of the REAL EnVision Detection System. Slides were counterstained with hematoxylin. The stained slides were digitalized using the ImageAccess 9 Enterprise software. Significance was calculated using the t test for paired samples. P 0. 05 was regarded as significant.

Results Panobinostat inhibits DNMT activity and expression in vitro After only 6 h of treatment, incubation of HepG2 and Hep3B cells led to a rapid and significant decrease in total DNMT activity by 46. 7% and 47. 4%, respectively. At later points in time, DNMT activity was stably reduced by approximately 20% in both cell lines, except for the 24 and 72 h time point in GSK-3 HepG2, where an in hibition of more than 40% was observed. Expression of DNMT1, DNMT3a and DNMT3b were then investigated by quantitative real time RT PCR.

NHBE cells at passages 2 to 4, and 16HBE cells were trypsinized and seeded onto the Costar Transwells inserts with 0. 4 um pore size at a density of 1. 5 �� 105 cells cm2 in media comprised of 50% BEBM and 50% DMEM F12 low glucose supplemented with the growth factors provided in the SingleQuot kits and retinoic acid. Once the cells reached confluency, they were switched to an air liquid any other enquiries interface for an additional 2 weeks to achieve mucociliary differentiation. PCN or IL 13 was added to the Transwell chambers for 24 hr. Sterile water was used as the control. NHBE cells were stained with mouse anti MUC5AC monoclonal antibody, and visualized with Alexa Fluor488 conjugated sec ondary antibodies under a confocal micro scope. Nuclei were stained with DAPI.

Brightfield and fluorescence images of these cells can be found in the Additional file 1, Figure S1 and Additional file 2, Figure S2. ROS assays ROS levels in PCN exposed NCI H292 cells were deter mined using the OxiSelect In Vitro ROS RNS Assay Kit according to the manufacturer protocols. The assay uses the spe cific ROS RNS probe dichlorodihydrofluorescin DiOxyQ. The DCFH DiOxyQ probe is first primed with a quench removal reagent, and subsequently stabilized in the highly reactive DCFH form. ROS and RNS species react with DCFH, which then rapidly oxidizes to to degradation with cell lysates. The amounts of mucins in total cell lysates were determined by west ern blotting using specific antibodies against MUC5AC and MUC5B or by ELISA kits. These ELISA kits have been previously used in mucin studies.

Posttranslational modification of FOXA2 Nuclear proteins from PCN stimulated or control NCI H292 cells were purified using the NE PER Nuclear and Cytoplasmic Extraction Reagents. FOXA2 was immunoprecipitated using anti FOXA2 antibody immobilized on Protein A G Agarose. Posttranslational modifications of FOXA2 were analyzed by western blot using antibodies against nitro tyrosine, acetylated lysine, methylated lysine, and ubiquitin. Neutralization of PCN by GSH NCI H292 cells were pretreated with GSH at indicated concentrations for 60 min before expos the highly fluorescent 2, 7 dichlorodihydrofluorescein. Fluorescence intensity is proportional to the total ROS RNS levels within the sample. The DCFH DiOxyQ probe can react with hydrogen peroxide, peroxyl radical, nitric oxide, and peroxynitrite anion, allowing for measurement of the total free rad ical population within a sample.

Mucin analysis NCI H292 or 16HBE cells were stimulated with indicated concentrations of PCN for 24 hr. Cells were lysed by the M PER Mammalian Protein Extraction Reagent in the presence of the Halt Protease Inhibitor Cocktail. The protease inhibitors were incorporated because of prior reports of sensitivity of the anti mucin Batimastat antibodies ure to PCN or sterile H2O for 24 hr.

To date, neuroblastoma, melanoma, Myc sellekchem driven lymphomas, leukemia and lymphoma cell lines and Fanconis Anemia cells have been identified to be Chk1 kinase dependent. This suggests that DNA repair, DNA damage response or DNA replication may be suitable candidates for single agent Chk1 inhibi tor therapy. Spontaneous triple negative breast cancer shares many of the characteristics of tumors derived from patients carrying mutations in the BRCA gene. BRCA is known to be involved in a variety of DNA repair pathways such as homologous recombination and base excision repair and are exquisitely sensitive to inhibitors of poly polymerase. Recent reports have demons trated the sensitivity of TNBC to PARP inhibitors as well as DNA damaging cytotoxic agents such as gemcitabine and cisplatin.

TNBCs have been shown to have reduced expression of DNA repair genes involved in base excision repair, nucleotide excision repair and the Fanconis Anemia repair pathways. This suggests that like cancers that arise in BRCA mutation carriers, spontaneous TNBCs may harbor underlying defects in DNA repair and we hypothesized that this cancer sub type may be a suitable candidate for single agent Chk1 inhibitor therapy. This hypothesis was substantiated in a screen of 26 solid cancer cell lines where TNBC cancer cell lines were among the most sensitive to growth inhibition by the novel, selective Chk1 inhibitor V158411. Even though V158411 is an extremely selective inhibitor of Chk1, it is difficult to ascertain the absolute selectivity of any small molecule kinase inhibitor.

To confirm this observation, two structurally unrelated Chk1 kinase inhibitors PF 477736 and AZD7762 potently inhibited the proliferation of TNBC breast cancer cell lines compared to two ER positive cell lines suggesting that the anti proliferative effects observed were due to Chk1 inhi bition and not the inhibition of an off target kinase. However, the sensitivity to the Chk1 inhibitors was not just limited to the TNBC cell lines as the HER2 postive, ER negative SKBr3 breast cancer cell line and the SKOV 3 ovarian cancer cell line were among the most sensitive cell lines to Chk1 inhibitor induced cell death. These results have recently been confirmed by another study demonstrating that four TNBC cell lines had reduced viability following Chk1 knockdown with a specific siRNA.

Additional studies have demonstrated that the Chk1 inhibitor AZD7762 synergistically combined with numerous PARP1 inhibitors to inhibit the growth of mammary carcinoma cells in vitro and in vivo. In these studies, AZD7762 demonstrated little sin gle agent activity in the breast cancer cell lines at the V158411 GSK-3 3. 1 1. 4 concentration tested. Of the cell lines tested in these pa pers, only two overlapped with our study. In light of these results, we attempted to understand the mechanism by which single agent Chk1 inhibitors induced TNBC and ovarian cancer cell death.

The cleavage of XIAP and the down regulation of Bcl xL and survivin observed after 8 h of treatment with TRAIL and SAHA was stronger than after 16 h with TRAIL alone. Similar results were selleck chemicals obtained with TRAIL and NaB. Moreo ver, the kinetic of inactivation of anti apoptotic proteins is similar to that of caspases activation by proteolytic cleav ages. We have previously shown that the down regulation of XIAP, Bcl xL and RIP induced by TRAIL and chemothera peutic drugs was caspases dependent. To analyse if the reduction of the anti apoptotic proteins XIAP, Bcl xL, survivin, and RIP induced by co treatments with TRAIL and HDACIs is likewise caspases dependent, SH EP cells were treated with TRAIL and NaB, SAHA or TSA in the presence of the pan caspases inhibitor zVAD.

As shown in figure 5d, the cleavage of XIAP, and the inactivation of Bcl xL, survivin, and RIP were protected by the addition of zVAD. Similarly, the activation of Bid and cas pase 3 induced by TRAIL and HDACIs were protected by zVAD. These results indicate that sensitisation to TRAIL by low doses of HDACIs increases the caspases dependent cleavages and inactivation of anti apoptotic proteins. In addition, the amount of BimEL was increased in the presence of zVAD indicating that the down regula tion observed by combined treatment was also mediated by caspases dependent cleavage. Down regulation of survivin sensitise NB cells to HDACIs Survivin is over expressed in most human cancers includ ing NB and was shown to be involved in inhibition of apoptosis in tumour cells.

In addition, down regula tion of survivin by siRNAs was shown to sensitise NB cells to TRAIL induced apoptosis. As survivin steady state level is reduced by co treatment with TRAIL and HDACIs, we explored its participation in HDACIs mediated sensiti sation to TRAIL. In this aim, survivin expression, was down regulated in SH EP cells by RNA interference using survivin siRNAs. As shown in figure 6a, survivin expression level was significantly reduced by survivin siRNA as compared to cells transfected with lipofectamine2000 or control siRNA. The effect of sur vivin silencing was analysed by proliferation assay. Results show that reduction of survivin expression with 100 nM siRNAs sensitised NB cells to TRAIL alone, to low doses of HDACIs as well as to combined treatments with weak doses of TRAIL and HDACIs.

Similar results were observed with 25 nM of survivin siRNAs. These results Entinostat indicate that down regulation of survivin induced by HDACIs and TRAIL may account at least in part to the sensitising effect of HDACIs to TRAIL induced cell death. Discussion The present study demonstrates that simultaneous admin istration of TRAIL and subtoxic doses of HDACIs strongly potentiates the triggering of apoptotic cascade in NB cells.

The normalized sensitivity coefficients for NF B acti vation were solved and plotted as heat maps to illustrate the dynamic relationship Rapamycin cost between the signaling compo nents and the system response. The sensitivity results clearly show that the NF B response is nearly completely insensitive to variations in some rate parameters, but also moderately or highly sensitive to others, consistent with earlier results which found that only a relatively small number of network parameters signifcantly influenced NF B activity. A notable feature of our analysis is that, with the excep tion of the NF B nuclear shuttling rates for which the sensitivity scores remain high throughout the entire response, NF B activity exhibits highly dynamic sensitivity with respect to most other para meters.

In other words, there is a strong temporal com ponent to the regulation of NF B activity, where variations in different parameters can exhibit great influ ence over certain phases of activity but have only mar ginal effects on activation during other time intervals. The first 20 min of NF B activity is predominantly influenced by the rates for IKK induced phosphorylation, ubiquitina tion and degradation and also IKK activation, with little contribution from the feedback parameters. As I Ba is degraded and free NF B ascends towards its maximal activity, the nuclear shuttling rates of free NF B have the greatest effect. However, the system shows extreme sensitivity to rates controlling the inner and outer feedback loops.

The system is very senstive to the rates for induced I Ba synthesis and its association with NF B during a time period coinciding with the decline of the first peak, with synthesis and binding rates negatively affecting NF B activation. The rate of conversion of inactivated IKK back to native IKK also is among the most signifi cant parameters in the attenuation of NF B activity. While NF B activity is at its lowest levels between 60 90 min, the stability of the remaining I Ba transcripts and the induced phosphorylation, ubiquitination and gradation of I Ba exert more influence on free NF B levels. The Anacetrapib second peak of NF B activity is regulated greatly by the nuclear import rate of free I Ba, as evi denced by the high sensitivity of ki3a only during this time period. Feedback from I Ba again has highly sig nificant contributions to the dynamics of the second peak, with induced synthesis of I Ba and its affinity to unbound I Ba having very high sensitivities. The NF B response is also highly sensitive to the outer A20 feedback loop in a time dependent manner. The rates for IKK inactivation by A20 significantly affect the termination of initial NF B activity as well as the second phase of activity.

This is highly signifi cant and can be quantified with a simple Fisher exact test. Explicitly, the probability p of 20 or more resistant samples in the top 25 correlations is less than 9 10 7. The K S significance score selleck catalog can be calculated by counting the number of times a random rearrangement of the samples gives a better enrichment, we find p 3 10 6. The enrichment plot is given in Figure 4A. As expected the top scoring correlations were dominated by samples from blood derived cells, for simplicity we restricted our analysis to the top 100 most significantly correlating sam ples. However, two studies in unrelated tissue pathologies were highly correlated with the corticosteroid resistant profile. These were a comparison of lung epithelia with cancer in smokers and a differential expression between healthy and cancerous pancreatic tissue.

The smoking study consisted of non diseased lung epithelia from 187 individual smokers 97 of whom were diagnosed with lung cancer. Ranking the samples accord ing to query correlation score we find that in the top 97 there are 64 cancer cases and in the bottom 90 there are 57 non cancer cases, with a significance score of p 5 10 5. The K S significance is p 2 10 4. The enrichment for positive correlations with the corticosteroid resistance profile in the cancer cases is shown in Figure 4B. Interest ingly, it has been shown that there is a down regulation of the glucocorticoid receptor in small cell lung cancer and reversing this promotes cancer cell apoptosis.

The pancreatic cancer study sought to establish a transcriptional signature of tumour versus normal pan creatic tissue by laser capture of cancerous and normal tis sue from the same pancreas. In total 39 sample pairs were published and we find a high positive correlation with the corticosteroid resistance profile, p 2 10 6 and a K S significance score of p 3 10 7. The enrichment curve is shown in Figure 4C. In this context it has been reported that loss of GR Carfilzomib expression has been seen in pancreatic car cinoma relative to normal tissue and elevating GR expression has been shown to inhibit pancreatic tumour growth in a hamster model. The query results are given in the additional file 2. Neurodegenerative Disease The analysis of gene expression changes associated with neurodegenerative disease has been hampered by the difficulty of extracting high quality RNA from post mor tem tissue. One way of validating a disease asso ciated gene expression profile is to show that it shares significant features with profiles derived from indepen dent experiments on related pathologies. A positive result would validate the query profile and furthermore lead to a more robust core response profile based on multiple experiments.

The lack of grapevines with F35H loss of function genotypes could be explained either by selection, which acted against knockout mutations, or by gene redun dancy, which obscured the effect of single gene loss silencing. The observation that our site an absence of 35 OH anthocyanins is generally tolerated in plants disfavours the first hypothesis. Furthermore, gene redundancy of F35Hs is commonplace in grape genomes, con trasting with most other species that have single or two copy F35Hs, or none at all. We have previously shown that F35Hs are highly duplicated, with multiple copies arrayed in clustered contigs of the Cabernet Sauvignon physical map. The genome assembly of the nearly homozygous line PN40024 allows a deeper investi gation into the structure of the F35H locus and into the evolutionary events that caused their proliferation in grapevine.

Expansion of gene families is common in plant gen omes, and results from various mechanisms of duplication, whole genome duplication, segmen tal duplication, tandem duplication, and transpositional duplication. WGDs have repeatedly occurred over evolutionary time in the common ancestor of eudi cots and in specific lineages. Segmental duplica tions occur over chromosomal regions, which may undergo subsequent rearrangement. Tandem duplica tions generate nearby gene copies. Small scale duplications may also cause transposition of one of the duplicate genes to an ectopic site. In this paper, local duplications of small fragments containing a single gene are referred to as tandem duplications. Duplication of DNA blocks 10 kb are referred to as segmental duplications.

Retention of duplicate genes results from a stochastic process, in which the effect of the earliest mutation occurring after duplication governs the fate of extra copies. Deleterious mutations occur much more fre quently than mutations resulting in novel and favourable functions. Following this assumption, gene disrup Brefeldin_A tion would largely prevail, with genomes populated by vestiges of ancient duplicates. This raises the question as to why intact duplicates are maintained and expressed much more frequently than expected by chance. Accord ing to the duplication degeneration complementation model, degenerative mutations promote pre servation of duplicate genes. Deleterious mutations in regulatory regions could eliminate different cis elements in either duplicate, making both copies necessary to pro vide the full complement of the expression profile of the ancestral single copy. This kind of partitioned expression among duplicate genes is referred to as sub functionalisation, and includes differential expression among organs and developmental stages, or in response to environmental cues.

At this stage of culture, HSCs show phenotypic features of fully activated find more HSC/MFs and a profile of cell surface mark ers identical to that of interface MF described in fibrotic and cirrhotic human livers. HSC/MFs were plated to obtain the desired subconfluence level and then incubated for 24 hours in serum free Iscoves medium in order to obtain cells at the lowest level of spontaneous proliferation before the addition of the dif ferent stimuli. Western blot Cells were lysed with 50 mM 1 piper azineethanesulphonic acid buffer pH 7. 5, 150 mM NaCl, 10% glycerol, 1% Triton X 100, 1. 5 mM MgCl2, 1 mM ethylene glycol tetraacetic acid, 10 g/ml leupeptin, 10 g/ml aprotinin, 1 mM phenylmeth ylsulphonyl fluoride and 100 mM sodium fluoride for 20 minutes at 4 C.

Cells were scraped from dishes and cen trifuged at 15,000 g for 20 minutes at 4 C. Supernatants were loaded for sodium dodecyl sulphate polyacrylamide gel electrophoresis gel. After transferring the proteins, blots were incubated with the desired primary antibodies and then incubated with peroxidase conju gated anti mouse or anti rabbit immunoglobulins in Tris buffered saline Tween containing 1% non fat dry milk and developed with ECL reagents or IMMOBILON Western reagents according to the manufacturers instructions. Akt activity An immune complex kinase assay of Akt activity was per formed as described elsewhere. Briefly, 100 mg of proteins were immunoprecipitated with anti Akt antibod ies followed by adsorption to protein G agarose.

Immu noprecipitates were then collected by a brief centrifugation and washed three times with washing buffer, 40 mM NaCl, 50 mM NaF, 1 mM ethylenediaminetetraacetic acid, 1 mM EGTA, 0. 5% Nonidet P 40, 20 mM b glycerophos phate, 0. 5 mM sodium orthovanadate, 1 mM phenyl methylsulphonyl Brefeldin_A fluoride, 10 mg/ml leupeptin, 10 mg/ ml pepstatin and 10 mg/ml aprotinin. The assay was per formed by resuspending the beads in kinase buffer, 100 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 10 mM b glycerophosphate and 0. 5 mM sodium orthovanadate in the presence of 1 mM protein kinase A inhibitor peptide, 50 mM unlabelled ATP and 6 Ci of ATP, using exogenous histone H2B as the substrate and incubating for 20 minutes at room temperature. Reaction products were run in a 12% SDS PAGE, stained with Coomassie Blue and visualised by autoradiography.

Evaluation of apoptosis Evaluation currently of cell apoptosis was performed by evaluation of PARP and caspase cleavage on Western blot. Statistical analysis All Western blots were representative of at least three to four experiments with similar results. Statistical analysis was performed by students t test. P values 0. 05 or 0. 01 were considered significant. Results In the first set of experiments we investigated the IGF I intracellular signalling downstream of PI 3K activation. As shown in Figure 1, IGF I induced phosphorylation of c Akt on Ser 473 residue after 15 minutes of incubation.