Decrease of wild type p53 protein might be due to the regulation of HDAC inhibitors on p53 transcription. Peltonen et al. dis covered that TSA stabilized wild type p53 in melanoma cell lines, but p53 protein accumulation was overridden by simultaneous downregulation of p53 mRNA, leading to a decrease in p53 protein. The mechanisms of p53 acetylation on both wild type and mutant proteins selleck chem inhibitor in dif ferent tumors after various HDACi exposure requires fur ther investigation. The Akt pathway plays an important role in cell growth, and its activation is common in tumors. Inhib ition of overphosphorylated Akt is a promising target ther apy in colorectal cancer . We observed pAkt overexpression in all three cell lines and subsequent downregulation after TSA treatment. A similar phenomenon was reported in other studies.

Chen et al. demon strated that HDACi caused Akt dephosphorylation in U87MG glioblastoma and PC 3 prostate cancer cells by disrupting HDAC protein phosphatase 1 complexes. LBH, another HDACi with a chemical structure similar to TSA, mediated Akt dephosphory lation in DLBCL DHL 6 cells through increased bind ing of PP1 to Akt. We further studied the downstream targets in the Akt pathway. Upregulation of p21 was previously commonly reported, with less data on p27. Repression of cyclin D1 from HDAC inhibitors was reported in mantle cell lymph oma. In our study, we found more significant al terations of p27 and cyclin D1 than p21 after TSA treatment. Both p21 and p27 were upregulated, and cyclin D1 was downregulated with decreasing expres sion of pAkt, which may account for the eventual cell cycle delay.

TSA also induces cell apoptosis in LY1 and LY8 cells. Bcl 2, an anti apoptosis regulator, was found to be downregulated after TSA treatment in LY1 and LY8 cells. In normal germinal centers, Bcl 2 is usually inactivated, rendering centroblasts and centrocytes susceptible to apop tosis. Abnormal retention of Bcl 2 leads to cells that do not die, thereby predisposing cells to malignant transformation. In our study, western blot analysis showed that the repres sion of Bcl 2 occurred at the translational level in LY1 and LY8 cells after TSA treatment. Its downregulation may be the combined effect of Akt dephosphorylation and p53 acetylation caused by TSA. However, Bcl 2 alteration in DoHH2 cells was quite different with LY1 and LY8 cells.

Bcl 2 gene rearrangement was previously reported in DoHH2, LY1 and LY8 cells. However, there is no detailed information regarding Bcl 2 amplification in the li terature. Our unpublished data showed that all three cell lines do not have apparent Bcl 2 gene amplification. One reason for the differential effects on Bcl 2 may be due to different levels of p53 acetylation. Low p53 acetylation Dacomitinib may contribute to DoHH2 cells resistance to apoptosis after TSA treatment at IC50.

Triglyceride Content Cells were incubated with 250M of IGOB131 for 72 h at 37 C in a humidified 5% CO2 incubator. Cells were col lected and lysed in lysis buffer. The total triglyceride content in cells was determined using a commercial triglyceride check details assay kit. The protein concentration was determined by using a BioRad DC pro tein assay kit. Inhi bition was expressed as percent decrease in triglyceride content against control. Glycerol 3 Phosphate Dehydrogenase Activity 3T3 L1 adipocytes were harvested 8 days after the initia tion of differentiation and were incubated with 250M of IGOB131 for 72 h at 37 C in a humidified 5% CO2 incu bator. Cells were washed twice with ice cold PBS on 3T3 L1 adipocytes, and lysed in 25 mM Tris/1 mM EDTA, pH 7. 5 for the measurement of glycerol 3 phosphate dehy drogenase specific activity.

G3PDH activity was determined according to the procedure of Wise and Green. Protein concentration was determined by the Bio Rad DC protein assay kit using bovine serum albumin as a standard. Enzyme activity was expressed as units of activity/mg protein. Inhi bition was expressed as percent decrease in G3PDH activity against control. Western Blot Assay Cells were incubated with 0 250M of IGOB131 acids for 12 and 24 h at 37 C in a humidified 5% CO2 incuba tor. They were collected and lysed in ice cold lysis buffer, 2 mM EDTA, 500M sodium orthovanadate, 1% Triton X 100, 0. 1% SDS, 10 mM NaF, 10g/mL leupeptin and 1 mM PMSF. The protein con centration was estimated with the Bio Rad DC protein assay using bovine serum albumin as a standard.

Total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 12% polyacryla mide gel. The proteins in the gel were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk in PBST for 1 h. Membranes were incubated with primary anti body at 4 C overnight and then with secondary antibody for 1 h. Membranes were washed in PBST for 10 min three times between each step. The signal was detected using the Amersham ECL system. The relative expression of PPAR, adiponectin, and leptin in 3T3 L1 adipocytes was quanti fied densitometrically using the software LabWorks 4. 5, and calculated according to the reference bands of actin. Statistical analysis Values are expressed as mean S. E. For multiple compari sons, a one way analysis of variance was used.

When ANOVA showed significant differences, post hoc analysis was performed with the Newman Keuls multiple range test using SPSS. Results Effect of IGOB131 on the inhibition of Intracellular Triglycerides and G3PDH activity Anacetrapib in 3T3 l1 adipocytes The effect of IGOB131 on percent intracellular triglyceride and G3PDH levels were evaluated as indicated in the method section and the results are presented in Table 1. The reported values are the means SD of three samples. For this study cellular harvesting and incubation was accomplished with IGOB131 as previously described in the method section.

In a COPD model of LPS induced airway inflammation and remodeling in guinea pigs, tiotropium abrogated the selleck chemicals Imatinib LPS induced in crease in neutrophils, goblet cells, collagen deposition and muscularised microvessels, but had no effect on em physema. These results suggested that endogenous acetylcholine plays a major role in the pathogenesis of this disease. EMT takes center stage as the convergence point between inflammation and airway diseases. Inflammatory mediators are known to induce downregulation of epithe lial cell cell adhesion and promote mesenchymal gene ex pression both in vitro and in vivo, and consequently inflammatory mediators have emerged as decisive factors in EMT induction. Although a number of molecules in volved in ACh mediated airway inflammation and remo deling have been identified, little is known regarding the role of ACh in EMT.

In the current study, we explored whether TGF B1 induced EMT can be influenced by non neuronal choli nergic system in lung epithelial cells, and if so, whether mAChR activation has similar effects to TGF B1 in EMT induction. Moreover, which of the two main pathways, the Smad pathway or the ERK pathway, both of which can be activated by mAChR agonists, was involved in EMT in lung epithelial cells. Methods Cell culture and treatment The human alveolar epithelial cell line A549 and human tracheobronchial epithelial cell line 16HBE were obtained from the American Type Culture Collection, and were maintained in Hams F 12 medium or Dulbeccos modi fied Eagles medium /HIGH Glucose supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator with 5% CO2 at 37 C.

Cells were grown in 96 or 6 well plastic tissue culture plates until confluence and then transferred into serum free medium containing 0. 1% FBS for 24 h. After that, cells were treated as designed. Western blot analysis Cells were lysed in RIPA lysis buffer and a protease inhibitor cocktail on ice for 5 min and scraped into a centrifuge tube. The lysates were centri fuged at 13,000 g for 5 min at 4 C. Total protein was mixed with sodium dodecyl sulfate sample buffer and heated at 100 C for 5 min. Equal amounts of samples were separated by 10% SDS polyacrylamide gel electro phoresis and transferred to polyvinylidene fluoride mem branes, which were then blocked with 5% non fat milk in TBS T for 1 h at room temperature and then incubated with primary antibodies overnight at 4 C.

After washing the membranes three times for 5 min each with TBS T, they were incubated with horseradish peroxidase conjugated secondary antibodies for 1 h at RT, followed by an additional three washes for 5 min each with TBS T. Bands were subsequently visualized on film using enhanced chemiluminescence reagents. Results were expressed relative to glyceraldehyde 3 phosphate dehydrogenase Drug_discovery band density used as a loading control.

To gain further insight into the mechanisms by which histone citrullination may regulate gene activity, we also tested whether Cl amidine affected levels of di methylated histone H3K9, a modification screening libraries closely asso ciated with transcriptional repression. We found that levels of this modification were not significantly affected by Cl amidine treatment. To validate the specificity of the acetylated histone anti bodies, the embryos were also treated with the HDAC inhibitor Trichostatin A, and as expected, staining for acetylated histones was elevated following TSA treatment. Taken together, these results suggest that in hibition of PADI mediated histone citrullination sup presses histone acetylation.

Histone hyperacetylation promotes histone citrullination in early embryos To further explore the potential interplay between histone citrullination and acetylation, the colocalization of citrulli nation at H3R2 8 17 and acetylation at H3K9 was tested in 2 cell embryos. Results revealed that these two histone modifications localize, in part, to different regions of the nucleus, and they do appear to colocalize at specific foci in 2 cell embryos. To further test whether there was a potential crosstalk between his tone citrullination and acetylation, levels of H3R2 8 17 citrullination were examined in embryos treated with either TSA or Cl amidine. To perform this experiment, PN zygotes were recovered from B6D2F1/J females and cul tured in KSOM medium supplemented with either 100 nM of TSA or 250 uM of Cl amidine, respectively, for 68 hours.

Results showed that, as expected, levels of H3Cit2 18 17 were significantly reduced following Cl amidine treatment. Interestingly, however, we found that induction of histone hyperactylation via TSA treatment also resulted in significantly increased levels of citrullination at H3R2 8 17. This observation further suggests that there is a reinforcing relationship between acetylation and citrullination and further highlights the po tential interplay between these two modifications on chro matin in preimplantation mammalian embryos. Conclusions This report is the first to document the presence of citrullinated histones in mammalian oocytes and pre implantation embryos. The use of three site specific citrullinated histone antibodies found that histone citrullination is likely playing several unique, yet to be defined roles on chromatin templated events.

We found that the PADI inhibitor, Cl amidine, potently blocks embryonic Brefeldin_A development beyond the 4 cell stage, thus further highlighting the important role of PADIs in early development. This observation also raises the possibility that PADI inhibitors could po tentially be utilized as novel contraceptives. Our study also showed that Cl amidine specifically suppressed histone acetylation on the H3 and H4 tails while not affecting levels of the transcriptionally repressive his tone H3K9 dimethyl modification.

When appropriate, a Newman Keuls Multiple Comparison Test was performed post hoc. Correlation analyses were performed using Pear sons coefficient of correlation. Significance was estab lished at p 0. 05. Values are reported as mean SD. Results Systemic and biologic response to CMV Arterial blood pressure was similar between the 4 groups. Blood pH, PO2 and www.selleckchem.com/products/Erlotinib-Hydrochloride.html PCO2 were maintained within the normal levels and were not different between the groups. Diaphragm in vitro contractile properties In the CMV group the force frequency curve shifted downwards when compared to C, as previously shown. In the MP 5 group, diaphragm force was further reduced compared to C and MP30. By contrast in the MP 30 group, diaphragm force was simi lar to that of C at all stimulation frequencies.

Tetanic tension was decreased with 30% after CMV when com pared to C and with an additional 15% in the MP 5 group while it was unchanged in the MP 30 group. Histochemistry Proportions of the different fiber types were similar between all groups. Compared to C, diaphragm CSA of the type IIx b fibers was significantly decreased with 29% after CMV, as previously shown, and with an addi tional 16% in the MP 5 group. CSA of the type IIa fibers were decreased in the MP 5 group only. In the MP 30 group CSA of the different fiber types remained unchanged and similar to that of C. Western blot analysis of calpain, calpastatin and caspase 3 Calpain activity, measured by talin degradation, was sig nificantly elevated after CMV, as previously shown, and to a similar extent in the MP 5 group when compared to C.

In the MP 30 group, talin degradation was similar to control levels. Calpastatin levels were significantly and similarly decreased after CMV and after administration of 5 mg kg MP compared with controls. In the MP 30 group, calpastatin expression was similar to that of the control group. Analysis of the caspase 3 mediated cleavage of aII spectrin revealed that CMV induced a significant rise in caspase 3 activity when compared to C. Caspase 3 activity was similarly increased in the MP 5 and the MP 30 group but this increase was significantly less compared to that of CMV. Significant negative correlations were Drug_discovery found between calpain activity and diaphragm force as well as with CSA of the type IIx b fibers. Significant positive correlation were observed between calpastatin and diaphragm force and calpastatin and CSA of the type IIx b fibers. 20S proteasome activity Compared to control, the chymotrypsin like activity of the 20S proteasome was increased by 48% in diaphragms from the CMV group. In contrast, both the low dose and high dose of corticoster oids prevented the CMV induced proteasome activation in the diaphragm.

Cells were seeded in 96 well plates and cultured under proliferating conditions and any other enquiries either tested during proliferation with or without hypoxia or were further used for differentiation studies. Subsequently differentiation was induced by withdrawal of the growth factors and cells were either incubated at 20% O2 or 3% O2 for an additional time period of 24 h and 72 h. The Wst 1 reagent was added to a final dilu tion of 1,10 for 2 h and the formazan produced by the metabolic activity of the cells was measured at a wave length of 450 nm using a plate reader. FACS analysis Cell cycle analysis For cell cycle analysis, proliferating or differentiating cells were harvested and fixed in ice cold 70% ethanol for 1 h at 20 C.

Prior to FACS measurement fixed cells were incubated with 1 mg ml RNase A for 30 min at 37 C following incuba tion with 50 ug ml propidium iodide for 30 min at 37 C. DNA content was measured by flow cytometry and analyzed by using the Cell Quest Pro software. Aggregated cells and debris revealed by for ward scattering were filtered out of the data set prior to analysis. To quantify G1, S, and G2 M populations, set tings for 2N and 4N peaks were defined within each experiment from the G1 S cells and applied to all sam ples within a given experiment. Antibody staining of neuronal proteins For the detection of bIII tubulin positive cells, cells were detached, centrifuged at 100g at room temperature, washed with PBS without Ca2 Mg2 and fixed with 1% PFA in PBS for 15 min. Then, cells were resuspended in washing buffer and stored at 4 C in the dark.

After centrifugation cells were resuspended in saponin buffer containing diluted mouse monoclonal FITC conjugated b III tubulin antibody and incubated for two hours at RT. Cells were washed twice with saponin buffer and resuspended in wash buffer for analysis. Mea surement was done using FACSCalibur in combination with Cell Quest Pro software. TUNEL assay and staining Apoptotic cells during differentiation were detected with an in situ cell detection kit. Detached cells were fixed with 1% PFA PBS for 15 min at RT. Afterwards cells were centrifuged and washing buffer was added. Until labelling, samples were stored at 4 C. For permeabiliza tion and labelling, samples were centrifuged and washed with PBS followed by an incubation with permeabiliza tion solution for two minutes on ice.

After an additional washing step with PBS, cells were incubated with TUNEL reaction mixture for Entinostat 1 h at 37 C at RT. As a positive control, cells were treated with DNase I for 10 minutes. As a negative control a sample treated with labelling solution was used. Subsequently cells were washed twice with PBS and a final volume of 250 ul PBS was added. The samples were measured and analysed with FACS Calibur and Cell Quest Pro Software. Western blot analysis For western blot analysis total cell extracts were pre pared.

Since the TiO2 Flowthrough and Wash fractions represent Pazopanib structure more than 70% of the sample and are highly complex, another fractionation step was performed. HILIC separation was used to reduce sample complexity, according to protein hydrophilicity. The raw data acquired from Thermo LTQ XL Orbitrap was converted to. mgf files and an in house MASCOT server was used to search for peptides containing dimethyl and carbamylation as a fixed modification and for phos phorylation in serine, tyrosine and threonine. The Thermo Proteome Discoverer software, version 1. 1 was used to quantify all peptides based on the total area of Extracted Chromatogram, and the absolute values were nor malized using a LOWESS algorithm. These data were input into the StatQuant software to evaluate the overall protein ratio by calculating the mean peptide ratio for all peptides corresponding to a given protein.

The list for all peptides and phosphopeptides quantified can be accessed in the Additional file 1, and a summary of upregulated and downregulated phosphoproteins in each experiment, sorted by period of time indutction with rhBMP2 is shown in Additional file 2. Phosphosite localization To assign phosphorylation sites, normalized Mascot delta score was used. Mascot delta score is the difference between the top two scores for the peptides identified by a given spectrum. Dividing this value by the score of the top score peptide, nor malized delta score is obtained. In order to have 1% FLR for correct phosphosite assign ment with 99% certainty, peptides with nMD score below 0. 36 were discarded.

A total of 950 unique phosphosites with 99% certainty that the sites were assigned correctly were iden tified. These sites were found on 235 different proteins and their distributions were 87. 5%, 11. 5% and 0. 8% for pS, pT and pY, respectively, which is comparable to previous works for mammalian cell types. All validated phospho sites with their MD scores are listed in Additional file 1. Phosphorylation motif database search The analysis carried out to determine which kinase could possibly be involved in phosphsorylation of a given phosphorylated residue from phosphoproteome data was performed using the NetworKIN site. Figure 4 shows a summary of the complete dataset represented by a graph containing kinase motifs occurrencies.

Network analysis using the ingenuity pathway analysis software In order to evaluate possible intracellular interactors with the phosphopeptides found, a network analysis was Carfilzomib performed. The Ingenuity Pathway Analysis software was used to map relation ships among proteins, distributed into different cellular compartments. From the total list of proteins found to interact with phosphoproteins, hits containing a transcription factor func tion were selected for further analysis of DNA binding motifs in osteoblast differentiation related genes.

By incorporating the drug target interaction data and sensitivities of training drugs with genomic signatures, we were able to achieve a cor relation coefficient of 0. 79 for prediction of Erlotinib sensi tivity using 10 fold cross validation. Carfilzomib 868540-17-4 The result illustrates the fundamental concept of the importance of drug target interaction and functional data under which we develop the sensitivity prediction method presented in this paper. By developing a framework around the functional and tar get information extracted from the primary tumor drug screen performed by our collaborators, we seek to develop a cohesive approach to sensitivity prediction and com bination therapy design. This necessitates the generation of the tumor pathway structure for individual patients to decide on the target inhibitors for therapy based on the personalized patient pathways.

We envision that the overall schematic of the design of personalized pathways and personalized therapy will be similar to the workflow shown in Figure 1. The explanations of the various steps in the design process are as follows, The primary contributions of this paper are, methods for extraction of numerically relevant drug targets from single run drug screens, design of the personalized TIM circuit based on drug perturbation data, algo rithms for sensitivity prediction of a new drug or drug cocktail, validation over canine osteosarcoma primary tumors and pathway flow inference using sequen tial protein expression measurements. The scope of the present article is concentrated around steps B, C and D of Figure 1.

The perturbation data required for our proposed method originates from a drug screen consisting of 60 small molecule inhibitors with quantified kinase interac tion behaviors. This drug screen, denoted Drug Screen Version 1. 0, consists of two sets of data, The first set is the experimentally generated drug sensitivities provided as 50% inhibitory concentration values. The IC50 values denote the amount of a drug required to reduce the population of cancerous cells in vitro by half. The sen sitivity values are expected to change during each new cell line tumor culture experiment. The generation of the sensitivities in step C can be done within 72 hours of ini tial biopsy using drug sensitivity assays which is a period of limited cell divisions for most primary cultures.

Thus, the estimated personalized maps may be closer to real time circuits in cancer cells akin to the signaling Anacetrapib found in an untreated patient within a day or two after biopsy, and not the evolving consensus pattern of signaling for grow ing and dividing tumor cells as subpopulations emerge with increased fitness in vitro. In addition, the drug screen contains experimentally derived half maximal con centration values for the interaction of each drug and each kinase target.

The gene ACAT2 is involved in cholesterol me tabolism, and expression of ACAT2 in cumulus cells is increased for infertile women as compared to fertile women. The gene HSD17B12 encodes for an en zyme that converts estrone to estradiol. It is also in volved in the synthesis Imatinib clinical of arachidonic acid and is essential for embryo survival in mice. Another gene related to DPR, HSD17B7, also converts estrone to estra diol and is essential for de novo cholesterol synthe sis in the fetus. In addition to genes involved in steroid synthesis, TSHB, a gene which codes for the B strand of the pituitary hormone, TSH, was associated with DPR. Thyroid function, which is under the control of TSH, can impact reproductive function in cattle. Some genes related to DPR may also affect re lease of neurotransmitters controlling hypothalamic pituitary function.

One, AP3B1, is involved in formation of synaptic vesicles, and APBB1 controls GnRH 1 neurogenesis. Another, TBC1D24, stimulates pri mary axonal arborization. Polymorphisms in TBC1D24 have been associated with shortened axons and epileptic seizures. Among the DPR genes involved in cell signaling are the G protein coupled receptors MRGPRF and MS4A8B, GPLD1, which cleaves cell surface proteins an chored by phosphatidylinositol glycans, the sialidase NEU3, which is important for insulin signaling, CACNA1D, a component of calcium channels, and DSC2, an important component of membrane rafts and cell cell junctions and which is involved in blastocoel formation. Similarly, OCLN is a major component of tight junctions and is involved in barrier stability.

An other gene involved in cell cell binding related to DPR is PMM2, which isomerizes mannose 6 phosphate into man nose 1 phosphate, which eventually is converted to GDP fucose and used to make fucosylated glycans. Fucosylated glycans serve several functions, including leukocyte endothelial adhesion, host microbe interactions, embryo compaction, and signal transduction. One gene associated with DPR, CSNK1E, is involved in paracrine regulation of cell function as a positive regulator of the canonical WNT B catenin pathway. The WNT pathway plays important roles in cell differentiation, preimplantation development, formation of the epiblast and implantation. Moreover, CSNK1E regulates circadian rhythm by controlling nu clear entry of PER1, a regulator of CLOCK.

Expres sion of PER1 was associated with depth of anestrus at the start of the breeding Drug_discovery season in beef cattle. Three genes related to DPR are involved with the function of spermatozoa in the female tract. The gene BSP3 aids in maintaining sperm motility during storage in the oviduct. Protein concentrations are associ ated with bull fertility and the mRNA is down regulated in the endometrium of heifers which carried a pregnancy to term compared to those in which the em bryo died after transfer.

As illustrated GSI-IX in Figure 1A, PDE6A, PDE6B, PDE6C, PDE6D, PDE6G and PDE6H mRNAs were expressed in the human lung. PDE6A, PDE6B, PDE6C and PDE6G showed no significant altera tions in the IPF lungs as compared to donor lungs. In con trast, PDE6D subunit was significantly down regulated in the IPF lungs as compared to the donor lungs and PDE6H showed a tendency of down regulation in the IPF lungs as compared to the donor lungs. In addition, the resultant PCR products were validated by direct sequencing, followed by BLAST analy sis that confirmed the similar sequence alignment for each subunit. Protein expression of the PDE6 enzyme subunits The protein content of the PDE6 subunits in whole lung tissue homogenates of donors and IPF patients was quan tified by immunoblotting.

As illustrated in Figure 2A, immunoreactivity was detected for PDE6A, PDE6B, PDE6D and PDE6G H subunits. PDE6A and PDE6B blocking peptide studies were carried out to reconfirm the specificity of PDE6A and PDE6B immunoreactivity. Additionally, pig retinal lysate served as a positive control for immunoreactivity and proper protein size. Notably, the PDE6D and PDE6G H subunits were significantly down regulated in the IPF lungs as compared to donor lungs, whereas PDE6A and PDE6B showed no significant alterations between donor and IPF derived lung tissues. Cellular localization of the PDE6 enzyme subunits The cellular localization of the PDE6 subunits was assessed by serial immunohistochemical stainings on tissue sections from donor and IPF lungs.

As shown in Figure 3A, PDE6A, PDE6B, PDE6D and PDE6G H were co stained with pro SPC, suggesting the presence of PDE6 subunits in ATII cells. PDE6A immunoreactivity was recognized in the cytoplasm and membrane of ATII cells, PDE6B immunoreactivity was recognized in the nuclei, PDE6D immunoreactivity in the cytoplasm and PDE6G H immunoreactivity in the membrane of ATII cells. PDE6 enzyme subunits expression in human AECs To confirm the AEC localization pattern, the PDE6 sub units were qRT PCR amplified from primary human donor and IPF derived ATII cells. All PDE6 subunits were found to be expressed by these cells. Notably, PDE6D mRNA levels were signifi cantly decreased in IPF derived ATII cells as compared to donor ATII cells. In contrast, PDE6A, PDE6B, PDE6G and PDE6H were not differentially regu lated in AECIIs from IPF versus control lungs.

TGF b1 down regulates PDE6D in A549 cells A549 cells were used as an in vitro AEC model. Firstly, the cells were characterized Anacetrapib for the expression of PDE6 subunits. mRNAs of all PDE6 subunits and the complete set of PDE6 pro teins were found to be expressed by these cells. Next, to explore whether TGF b1 promotes PDE6D down regulation in AECs, A549 cells were trea ted with two different concentrations of TGF b1 for 12 and 24 h.