OCT4, KLF4, SOX2, GLIS1 and c-MYC, robustly generated iPSCs from adult human fibroblasts aged 54 to 77 and from a 24 year old cardiomyopathy patient donor. Interestingly, 5F-srRNA induced LIN28A, which was one of the original reprogramming factors. 5F-srRNA also accelerated the generation of iPSCs by seven days compared to 4F-srRNAs. Further improvements include phosphatase treatment to remove 5' phosphate and use of Lipofectamine MessengerMAX that increased transfection efficiency to ~90%. Together, these improvements enabled us to efficiently generate iPSCs from human fibroblasts using 5F-srRNA while eliminating both puromycin selection and feeder cells.We previously devised a polycistronic, synthetic self-replicating RNA (srRNA) to generate human induced Pluripotent Stem Cells (iPSCs) that simultaneously expresses four reprogramming factors (4F). However, while the best 4F srRNA efficiently generated iPSCs from young fibroblasts, it was inefficient on adult human fibroblasts (>50 years). To increase the iPSC generation efficiency, we included additional reprogramming factors. We found that a single transfection of a five factor (5F) srRNA, containing  OCT4, SOX2, KLF4 and cMYC, or OCT4, SOX2, NANOG and LIN28 in mouse and/or human fibroblasts [c-MYC [Induced pluripotent stem cells (iPSCs) were originally generated by infection of retroviruses expressing roblasts –3, whichs [c-MYC . Therefos [c-MYC . Of thesin vitro. A single srRNA transfection resulted in extended expression of four reprogramming factors  in the presence of B18R protein. Because exposure of cells to single-stranded RNA, including srRNAs, induces a strong IFN-α/β innate immune responses [We previously developed an srRNA system for generating human iPSCs by expressing the reprogramming factors using the RNA replicon of Venezuelan Equine Encephalitis (VEE) virus . The VEEesponses , 13, 14,OCT4, KLF4, SOX2, GLIS1 and c-MYC (OKSi-GM), significantly increased iPSC generation in six different adult human fibroblasts, including old adult fibroblasts and a cardiomyopathy patient donor. Moreover, the time to reprogramming by 5F-srRNA was shortened by a week compared to 4F-srRNAs. Lastly, by improving srRNA quality with phosphatase treatment and using a new transfection reagent, Lipofectamine MessengerMAX, a single 5F-srRNA transfection resulted in ~90% efficiency of iPSC generation. Together, these improvements enabled the generation of iPSCs without the need for puromycin selection or feeder cells.Although 4F-srRNAs (OKS-iM and OKS-iG) worked well to generate iPSCs from both neonatal and adult human fibroblasts, the efficiency varied depending on the cell source, age of cells and/or four factor combinations. To address these problems, here we sought to increase the efficiency for iPSC generation by adding new factor(s) into the 4F-srRNA. We found that five factor srRNA (5F-srRNA), including BJ foreskin fibroblasts were obtained from ATCC and primary human foreskin fibroblasts (HFF) were kindly obtained from M. Haas (UCSD). FB#31 , FB#32 , and FB#33  fibroblasts were obtained from N.C. Chi (UCSD). HFB  human adult fibroblasts were obtained from L.S.B. Goldstein (UCSD). NHDFc  and HDF  human dermal fibroblasts were obtained from PromoCell (c-12302) and Cell Applications (106-05a), respectively. All fibroblasts were cultured in DMEM containing 10% FBS, MEM Non-Essential Amino Acids (NEAA), Pyruvate, penicillin, and streptomycin. HUES-9 and iPSCs were cultured with ES culture medium in Knockout D-MEM containing 20% Knockout SR, GlutaMAX, NEAA, 2-Mercaptoethanol, penicillin, streptomycin, and bFGF (10 ng/ml). Advanced DMEM containing 10% FBS, GlutaMAX, penicillin, and streptomycin was transiently used for iPSC reprogramming. STO feeder cells were prepared by mitomycin C treatment . Matrigel (BD Bioscience) or Laminin-521 coated wells and conditioned medium of STO feeder cells were used for feeder free culture.LIN28A (BC028566) cDNA was obtained from ATCC. LIN28A, cMYC and GLIS1 ORFs were cloned into T7-VEE-OKS by inserting the P2A sequence between them preceded by an IRES sequence yielding T7-VEE-OKS-iML, T7-VEE-OKS-iGL, T7-VEE-OKS-iGM and T7-VEE-OKS-iGML. PCR primers for cDNA amplification and P2A oligo sequences are listed in T7-VEE-OKS-iM, T7-VEE-OKS-iG and (T7-VEE-Gfp) were previously constructed as described . The LINRNA synthesis was performed as described . BrieflyCells were passaged on 6-well plate on day 0 and cultured to 80–100% confluency on day 1. To minimize the interferon response, cells were pretreated with 20% B18R conditioned media (BR-CM) for 20 min before transfection . Cells were transfected with 1 μg of each srRNA or co-transfected with srRNA and B18R mRNA  in the presence of 20% B18R-CM. After 3 hr, transfection medium was changed to the Advanced DMEM containing 20% B18R-CM. On day 7, Advanced DMEM was replaced to ES culture medium containing 20% B18R-CM. Where used, puromycin (0.8 μg/ml) was added from day 2 to 10. Cells were passaged onto STO feeder cells with several dilutions on day 10 and cultured in ES culture medium containing 20% B18R-CM. Culture media containing 20% B18R-CM were changed every day until iPS cell colonies were generated. For generating iPSC in feeder free condition, cells on day 10 were passaged to Laminin-521  or Matrigel  coated 6-well plate with several dilutions in STO feeder conditioned medium containing 20% B18R-CM and bFGF (10 ng/ml). STO feeder conditioned medium was prepared from one day culture of STO feeder cells in ES culture medium (without bFGF and B18R-CM), filtrated and store at -20°C until use. For puromycin-free selection, cells were passaged in STO feeder with 6 times dilutions in the Advanced DMEM containing 20% B18R-CM on day 5. ES culture medium was used from day 7. Rock inhibitor  was added into ES culture medium when cells were passaged in STO feeder or feeder free conditions.The maximum transfection efficiency with Lipofectamine MessengerMax  was obtained with serum free condition (data not shown). Cells were passaged on 6-well plate on day 0 and cultured to 50 or 100% confluency on day 1. Cells were washed once with DMEM (no antibiotics and no serum) and then added DMEM  prior to transfection. For serum-free srRNA transfection, we used B18R mRNA to suppress the IFN responses and omitted the 20% B18R-CM treatment. Transfection complex with 5F-srRNA (1 μg), B18R mRNA (1 μg) and mMax reagent (6 μl) for one well of 6-well were prepared according to the manufacture’s protocol except for omitting the 10 min incubation of diluted messengerMax reagent and transfected into cells in the absence of serum. No incubation of the diluted messengerMax is significantly affect on the transfection efficiency of srRNAs. After 3 hr incubation, medium was changed to the Advanced DMEM containing 20% B18R-CM. 4 hr incubation will increase transfection efficiency for mMax reagent in current protocol (data not shown). For puromycin-free condition, cells were passaged to Matrigel or STO feeder on day 3 or day 5 with 6 or 12 times dilutions. ES culture medium was used from day 5.Cells were lysed with 2x RIPA buffer containing 0.3 M NaCl, 80 mM Tris-HCl (pH 7.5), 0.4% SDS, 2% Triton-X 100, 2% sodium deoxycholate, 100 μg/ml phenylmethylsulfonyl fluoride (PMSF), aprotinin (5 μg/ml) and leupeptin (5 μg/ml). Equal amount of proteins (~20 μg) were used for 9% SDS-PAGE and electroblotted onto a Nitrocellulose membrane. Membranes were incubated with primary antibodies O/N at 4°C after blocking with 4% Milk in PBS-T (0.05% Tween-20), and incubated with horseradish peroxidase-conjugated anti-rabbit, goat or mouse IgG (Santa Cruz). Protein bands were visualized using the ECL reagent .Antibodies used in this research are as follows: anti-OCT4 (sc-9081), anti-KLF4 (sc-20691), anti-GLIS1 (sc-67584), anti-c-MYC (sc-42), TRA-1-60 (sc-21705), SSEA1 (sc-21702), SSEA4 (sc-21704), anti-mouse (sc-2005), anti-rabbit (sc-2004) and anti-goat (sc-2020) from Santa Cruz; anti-SOX2 (AF2018) and anti-NANOG (AF1997) from R&D Systems; anti-LIN28A (#46020) from Abcam; anti-TRA-1-81 (09–0011) from Stemgent; Alexa Fluor 488 anti-mouse (A11029), Alexa Fluor 488 anti-rabbit (A11034) and Alexa Fluor 488 anti-goat (A11055) for immunostaining of iPS clones from Life Technologies. IRDye800 anti-mouse (610-132-121) for Odyssey imaging of iPSC colonies from Rockland.2, pH 9.5) and stained with AP-Staining solution  in AP buffer for 15 min. For TRA-1-60, TRA-1-80, SSEA4 or SSEA1 staining, AP stained cells were then fixed in 4% paraformaldehyde for 10 min, and then treated with 0.1% Triton X-100 for 10 min. Cells were blocked with 2% BSA for 1 hr, and then incubated with primary antibodies in PBS at 4°C overnight. Cells were washed and incubated with IRDye 800 (1:800 dilutions) for 3 hr. Colonies were scanned with Odyssey imaging system (LI-COR).After the generation of primary iPSC colonies, cell were washed with AP buffer , and then incubated with primary antibodies in PBS at 4°C overnight. Cells were washed and incubated with secondary antibodies followed by incubation with Hoechst 33342, and then washed and stored in PBS. Primary antibodies such as rabbit anti-OCT4, goat anti-NANOG and anti-SOX2, mouse anti-SSEA4, anti-TRA-1-60 and anti-TRA-1-81 antibodies were used at 1:100 to 1:500 dilutions. Alexa Fluor 488 (BD Biosciences) secondary antibodies were used at 1:800 dilutions.2 = 0.999), and then amount of 5F-srRNA in total RNA was calculated.Total RNAs were isolated with TRIzol (Life Technologies). For RT-PCR detection of srRNA, cDNAs were synthesized with iScript cDNA synsethis kit (Bio-Rad) from 1 μg of total RNA. 2 μl of 20 μl RT reaction was used for PCR amplification. PCR was performed with Taq DNA plolymerase (NEB) supplemented with PCRx enhancer (Life Technologies): 3 min at 94°C for initial denature; 36 cycles of 94°C for 25 sec, 56°C for 25 sec, 68°C for 30 sec; followed by 72°C for 5 min. Primers used for RT-PCR are described before . For TaqTotal RNAs of iPSCs clones, HUES-9 and BJ were isolated with TRIzol (Life Technologies). TaqMan RT-PCR reactions were carried out using RNA-to-Ct one-step reaction (Applied Biosystem) according to manufacturer’s protocol. 10 ng of total RNA were used per reaction. Primers and probes were obtained from AB TaqMan Gene Expression Assay catalog . Quantitative PCR reactions were carried out in triplicate, and conditions were as followed: 20 min 55°C, 10 min 95°C, 40 cycles of 95°C for 0.15 min, 65°C for 1 min. Data were analyzed on the 7300 real-time PCR system (Applied Biosystems) using the delta-delta Ct method.iPSC clones were intramuscularly or subcutaneously injected into the hind limb muscles or dorsal flank of NRG mice (approximately 10 cm dish cultured cells for 1 injection). After 2 to 3 months of injection, tumors were dissected and fixed with 4% paraformaldehyde, embedded into paraffin, and sectioned for hematoxilin and eosin (H&E) staining.All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of University of California, San Diego (protocol number S01103). Studies using human ESCs and hiPSCs were approved by the IRB/ESCRO committee at University of California, San Diego (project number 071552ZF).LIN28A is frequently used to increase the efficiency of iPSC generation by mRNAs and episomal plasmids in combination with the Yamanaka Factors  [LIN28A Open Reading Frame (ORF) to the 4F-srRNA construct after cMYC or GLIS1 with an intervening self-cleavable P2A peptide to generate OKS-iML and OKS-iGL srRNAs, respectively  to generate iPSCs. To our surprise, we did not observe a significant increase in Alkaline Phosphatase (AP) positive colonies with the OKS-iML srRNA compared to 4F-srRNAs with only a <2-fold increase  into neonatal and adult human fibroblasts . Howevernd cMYC) –18. LIN2ectively . The OKSincrease . HowevercMYC, a master transcription factor that drives cell division and metabolism [cMYC and cMYC plus LIN28A with an intervening self-cleavable P2A peptide into the OKS-iG srRNA to generate OKS-iGM 5F-srRNA and OKS-iGML 6F-srRNA, respectively , 5F-srRNA (OKS-iGM), and 6F-srRNA (OKS-iGML). For iPSCs generation, BJ fibroblasts were transfected with each srRNA once, placed under puromycin selection and then passaged onto feeder cells. By day 7, both 5F-srRNA and 6F-srRNA transfected cells showed strong morphological changes, while only a small number of 4F-srRNA transfected cells showed signs of morphological change . By day We next compared the generation of iPSC colonies by 4F-srRNA, 5F-srRNA and 6F-srRNA with three adult human fibroblasts. FB#31 was a healthy 55 year old male donor, FB#32 was a healthy 54 year old female donor, and FB#33 was a 24b year old male cardiomyopathy patient donor. Similar to BJ cells, many large iPSC colonies were generated by 5F-srRNA transfection on day 21 . By day Given the efficiency of 5F-srRNA iPSC to generate iPSCs, we tested 5F-srRNA under feeder-free conditions. We observed many iPSC colonies in 5F-srRNA feeder-free conditions using Laminin-521 or Matrigel , Table 1To characterize iPSCs generated by 5F-srRNA, we isolated multiple colonies from 5F-srRNA transfections of BJ, FB#31, FB#32, and FB#33 fibroblasts. qRT-PCR analysis for expression of OCT4 (endogenous), SOX2 (endogenous), NANOG, LIN28A, TERT, DNMT3, and TDGF1 in each clone was comparable with HUES9 human ESCs . In addiDuring the 5F-srRNA and 6F-srRNA experiments, we detected differences of srRNA quality that were due to the 5' capping reaction. We used an enzymatic capping method and 2’-O-methyltransferase that generates a Cap-1 RNA with a theoretical yield near 100%. However, we observed varied transfection efficiencies depending on the capping reaction enzyme lot number from commercial venders. Poor capping efficiency leads to srRNAs contaminated with non-capped srRNA that contain a 5’-triphosphate that induces strong RIG-I and PKR innate immune responses , 19–22. In our hands, the transfection efficiency of GFP srRNA (~10 kb) into human fibroblasts using Lipofectamine 2000 is 40~60%, while the transfection efficiency of GFP mRNA (~0.9 kb) is > 90%. To improve iPSC generation efficiency, we tested a newly released transfection reagent, Lipofectamine MessengerMAX. Using Lipofectamine MessengerMAX we obtained ~90% transfection efficiency of the 10 kb GFP srRNA . LikewisMany integration-free methods, including episomal plasmids , 18, mRNOCT4, KLF4, SOX2, GLIS1, and cMYC was superior to both 6F-srRNA, that also contains LIN-28A, and our original 4F-srRNA (OKS-iM or OKS-iG) [Oct4, Sox2, Klf4, cMyc and Glis1) in mouse fibroblasts, but not in human fibroblasts, by introducing these factors with retrovirus infection. In human fibroblasts, they obtained similar iPSC colonies with OSKMG and OSKG, and both were better than OSKM. In contrast, 5F srRNA containing OCT4, KLF4, SOX2, GLIS1, and cMYC significantly increased the human iPSC generation as compared to both 4F-iM and 4F-iG srRNAs, especially in old adult human fibroblasts. Moreover, compared to retroviral infection with each reprogramming factor or transfection of individual mRNAs, 5F srRNA expresses each factor from a single polycistronic srRNA, so the ratio of reprogramming factors is constant in each cell. This is critical, because the expression ratio of reprogramming factors affects on the efficiency of human iPSC generation [cMYC inhibit the generation of iPSCs [To do so, we cloned in additional reprogramming factor(s) open-reading frames into the single srRNA and assayed for iPSC generation. We found that the five factor combination of  OKS-iG) . Previou OKS-iG)  reportedneration , 26. We of iPSCs . We alsoWe tested the ability of 5F-srRNA to generate iPSCs on six different older adult human fibroblasts from 24 to 77 years of age and obtained more than 100 iPSC colonies from a starting well . A parallel comparison of 4F-srRNA and 5F-srRNA showed that 5F-srRNA generated iPSC colonies 4-fold to >10-fold more efficiently than the 4F-srRNA . MoreoveLastly, during preliminarily experiments, we detected a low capping efficiency of srRNAs resulting in activation of an innate immune response. However, the addition of a phosphatase treatment step to remove the 5’-triphosphate of non-capped RNA successfully removed the non-capped RNA and solved this problem. Furthermore, because we observed some srRNA degradation during the poly A reaction step (data not shown), we built in a 150 base poly A tail into backbone srRNA vector to omit the poly A tail reaction. This improvement increased the srRNA quality, and resulted in a slight increase in the number of iPSC colonies (data not shown). Previously due to a poor transfection of the 16.5 kb 5F-srRNA, we performed a puromycin selection step to remove untransfected cells. However, using the new Lipofectamine MessengerMAX transfection reagent for long RNAs sufficiently increased the transfection efficiency and thereby allowed us to eliminate the puromycin selection. Notably we obtained similar number of iPSC colonies comparable to 5F-srRNA transfection with puromycin selection.In summary, a single transfection of 5F-srRNA efficiently generated iPSC colonies from six different adult human fibroblasts ranging from 24 to 77 years old, including a cardiomyopathy patient donor cells, seven days faster than our 4F-srRNA . SurprisWe generated a 5F-srRNA that worked faster and efficiently for generating iPSCs in all human fibroblasts tested including a cardiomyopathy patient donor. Moreover, by improving srRNA quality with phosphatase treatment and using a new transfection reagent, Lipofectamine MessengerMAX, a single 5F-srRNA transfection resulted in ~90% efficiency of iPSC generation. Together, these improvements enabled the generation of iPSCs without the need for puromycin selection or feeder cells.S1 FigA iPSC generation byh OKS-IM, OKS-iG, OKS-iML and OKS-iGL srRNAs in BJ cells. iPSCs were generated by co-transfection of srRNA plus B18R mRNA (1:1 ratio) in BJ and HFF cells. AP staining of iPS colonies performed on day 28. B Number of AP positive iPSC colonies per starting 6 well.(PDF)Click here for additional data file.S2 FigA qRT-PCR analysis of 5F-srRNA in BJ-5F-srRNA iPSC clones. "-" = BJ cells without sr-RNA (negative control); "+" = srRNA transfected BJ cells (positive control). BJ-5F-clones (1–8). iPSC clones were mechanically isolated and cultured in the absence of B18R. Total RNA was isolated from Passage 3 and 5, and used for qRT-PCR. Primers were in nsP2 region and Oct4-Klf4 (OK) region. B qRT-PCR analysis with TaqMan probe for srRNA (nsP1 region) was performed in BJ-5F-srRNA clones (1–8). * = no amplification.(PDF)Click here for additional data file.S3 FigA OCT4 expression with phosphatase treated 5F-sRNA. 5F-srRNA was prepared with or without phosphatase treatment. 5F-srRNA and B18R mRNA were co-transfected into BJ cells with Lipofectamine 2000 for Western blotting. 5F-Phos (-): no phosphatase treated 5F-srRNA; 5F-Phos(+): phosphatase treated 5F-srRNA. B TagGFP2 srRNA (gifted from EMD Millipore) was prepared with or without phosphatase treatment. TagGFP2 srRNA was transfected into BJ cells with MessengerMax reagent in the presence of recombinant B18R protein. One day after the transfection, GFP expression was measured by FACS. Phos (+): Phosphatase treated srRNA; Phos (-): No phosphatase treated srRNA. C Microphotographs of cells from (A). Scale bar, 250 μm.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Escherichia coli (EHEC) produce attaching and effacing (A/E) lesions, characterized by the formation of actin pedestals, on mammalian cells. A bacterial T3SS effector NleL from EHEC O157:H7 was recently shown to be a HECT-like E3 ligase in vitro, but its biological functions and host targets remain elusive. Here, we report that NleL is required to effectively promote EHEC-induced A/E lesions and bacterial infection. Furthermore, human c-Jun NH2-terminal kinases (JNKs) were identified as primary substrates of NleL. NleL-induced JNK ubiquitylation, particularly mono-ubiquitylation at the Lys 68 residue of JNK, impairs JNK’s interaction with an upstream kinase MKK7, thus disrupting JNK phosphorylation and activation. This subsequently suppresses the transcriptional activity of activator protein-1 (AP-1), which modulates the formation of the EHEC-induced actin pedestals. Moreover, JNK knockdown or inhibition in host cells complements NleL deficiency in EHEC infection. Thus, we demonstrate that the effector protein NleL enhances the ability of EHEC to infect host cells by targeting host JNK, and elucidate an inhibitory role of ubiquitylation in regulating JNK phosphorylation.As a major diarrheagenic human pathogen, enterohemorrhagic  Escherichia coli (EHEC) can cause attaching and effacing (A/E) lesions to form in the colons of animals and humans, contributing to severe bacterial infection. NleL, an E3 ubiquitin ligase from EHEC O157:H7 is one of the bacterial type III secretion effectors that may be involved in the regulation of A/E lesions. However, NleL’s exact host targets and the detailed mechanistic actions are still unclear. Here, we report that the effector protein NleL effectively promotes EHEC-induced A/E lesions and bacterial infection by targeting the host JNK protein. Specifically, we find that NleL-mediated JNK ubiquitylation abolishes phosphorylation and activation of host JNK, subsequently suppressing the host JNK/AP-1 signaling pathway to favor the formation of EHEC-mediated actin pedestals on the surface of mammalian cells. Collectively, our work has not only discovered the A/E lesion-promoting function of NleL during EHEC infection, but also revealed a novel regulatory mechanism of host JNK protein.Enterohemorrhagic  EHEC is a globally spread, pathogenic Escherichia coli that infects animals and humans . La. Lain viin vitro –18. Whilin vitro , NleL’s In this study, we have identified human JNK as the first substrate of the bacterial E3 ligase NleL. The JNK  family includes three highly homologous isoforms: ubiquitously expressed JNK1 and JNK2, and tissue-specific JNK3 . JNKs arnleL-deletion mutant (ΔnleL) was first constructed by chromosomally inactivating the nleL gene in the parental EHEC O157:H7 Sakai strain (RIMD 0509952) as described previously [nleL has no effect on the growth of EHEC in culture medium , but not the enzymatically-dead NleL mutant C753A (where the active site Cys at position 753 is replaced with Ala) (ΔnleL + pC753A), effectively restored the strong adherence of EHEC to host cells  system with full-length NleL as the bait. A cDNA encoding human JNK1, H screen . A serieH screen . A GST pin vitro . Compareanalyses ,17, fullnleL strain had little or no impact on JNK ubiquitylation. Moreover, complementation of ΔnleL with wild-type NleL (but not the C753A mutant) effectively promoted JNK ubiquitylation in the infected cells. Thus, NleL could induce JNK ubiquitylation in the EHEC-infected host cells.We next asked whether the interaction between NleL and host JNK might cause JNK ubiquitylation. As shown in 170-782 was sufficient to mediate the assembly of poly-ubiquitin (poly-Ub) chains in vitro [in vitro and in vivo  analysis ,25. MS ain vivo ubiquitylation assays were performed with JNK1 mutants bearing Lys-to-Arg substitutions at each potential ubiquitylation site. Single Lys-to-Arg substitution on each of three sites  markedly attenuated NleL-induced JNK1 poly-ubiquitylation , JNK1 was ubiquitylated efficiently but poorly phosphorylated, suggesting that NleL-mediated JNK1 ubiquitylation might adversely impact JNK1 phosphorylation . MoreoveJNK family proteins share over 90% sequence homology to each other , includiCurrently, two MAP2Ks, MKK4 and MMK7, are known to phosphorylate and activate JNK proteins, with MKK7 being more specific to JNK . It is nE. coli do not have the ubiquitin system, NleL should not conjugate Ub to JNK1 in bacterial cells. If NleL could mediate modifications in addition to ubiquitylation, it could still potentially modify and inactivate JNK1 when co-expressed with JNK1 in E.coli. However, we found that MKK7 readily phosphorylated purified JNK1 that was co-expressed with either GST-tagged NleL or GST alone in the E. coli BL21 (DE3) strain (Since ) strain . These dAltogether, we conclude that NleL-induced ubiquitylation at the K68 residue of JNK1 suppresses the phosphorylation of JNK1, through disrupting the JNK1-MKK7 interaction.bona fide physiological substrate of JNK, while the C753A mutant did not  [As c-Jun is a major component of the AP-1 transcription factor , we next (CCND1) . As expee.g. CD44, TPM1, ARPC1B and EZR) are known to be important for the formation of EHEC-induced pedestals. A protein-protein interaction (PPI) network analysis was performed to characterize the potential interplay among the actin-associated proteins targeted by AP-1 and the host proteins identified in EHEC actin pedestals  caused A/E lesions on 21-day-old Caco-2 monolayers, marked by microvillus damage. Complementation of the ΔnleL strain with wild-type NleL, but not the C753A mutant, restored the EHEC A/E lesion-forming ability; SP600125 treatment also rescued the ability of ΔnleL to form A/E lesions on the Caco-2 monolayer. These data suggested that the suppression of JNK1/2 functions in host cells could compensate for nleL-deletion-caused loss of the EHEC ability to colonize the host and form A/E lesions. JNK proteins are thus identified as the critical host targets for NleL to promote EHEC O157:H7 infection  could be used as an in vitro infection model to study A/E lesions. Caco-2 monolayers can mimic human colonic epithelium for EHEC infection, providing an alternative approach to in vivo infection.As described above, odentium –38. WhilDifferent Ub chain linkages have different impacts on targeted proteins  –41. HereNleL-mediated JNK mono-ubiquitination appeared to drastically suppress JNK activity, although the mono-ubiquitylated sub-population of JNK1 seemed limited . Currently there are two accepted explanations: 1) cellular proteins can exist in different subcellular populations, so targeted ubiquitylation of a particular subpopulation can be sufficient to generate significant impacts on a specific pathway; 2) NleL-induced JNK1 ubiquitylation should be a dynamic process balanced by removal of ubiquitin by deubiquitylating enzymes (DUBs), as there are plenty of DUBs in mammalian cells –45. In oThe JNKs are master regulators in mammalian cells . It is wWorldwide, outbreaks of EHEC O157:H7 infection constitute constant and serious threats to human population and live stocks, without effective treatments . AntibioAll animal use procedures were in strict accordance with the Guide for the Care and Use of Laboratory Animals , approved by the Institutional Animal Care and Use Committee (Protocol number SIBCB-S330-1512) of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.E. coli O157:H7 Sakai strain was inserted into pCDNA3.0 and pEGFP-C1 for mammalian expression, and pGEX-4T-1 for recombinant expression in E. coli. NleL DNA was also ligated into the pTRC99A vector for complementation in EHEC . Genes for encoding JNK1α1, JNK2α2 , JNK3α2 (Addgene #13759) and MKK7 (Addgene #14623) were cloned to pCDNA3.0 vector with a C-terminal Flag tag (or HA tag), and pET28a vector with 6× His tag. AP-1 luciferase reporter plasmid was a gift from Dr. Jine Yang . Plasmids expressing HA-tagged Ub and its mutants were described previously [DNAs for NleL amplified from the genomic DNA of eviously . All the6 tag (M0812-3), Proteintech for mouse anti-Cyclin D1 (60186-1-Ig), rabbit anti-Flag tag (20543-1-AP) and mouse anti-GAPDH (60004-1-Ig). Peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgG secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. Chemicals were purchased from Sigma if not otherwise indicated: PS-1145 (Santa Cruz Biotechnology), ATP (Thermo Scientific Fermentas). JNK inhibitor SP600125 (S1460) was from Selleck. HeLa, Caco-2 and HEK293T cells were obtained from the American Type Culture Collection (ATCC). All cell culture products were from Corning.Antibodies for JNK1 (2C6) (#3708), phospho-SAPK/JNK (81E11) (#4668), phospho-SAPK/JNK (G9) (#9255), phospho-c-Jun (54B3) (#2361), phospho-c-Jun (#9261), caspase-3 (#9662), VASP (9A2) (#3132) and phospho-VASP (Ser157) were obtained from Cell Signaling Technology. Anti-JNK2 antibody (EP1595Y) (ab76125), anti-MEK7 antibody (EP1455Y) (ab52618), anti-JNK1/2/3 antibody (ab179461) and anti-MEK7 (phospho S271 + T275) (ab4762) were purchased from Abcam. Rabbit anti-HA antibody (H6908), mouse monoclonal anti-Flag antibody (F1804), and Anti-Flag M2 affinity gel (A2220) were from Sigma. Other antibodies were purchased from BD pharmingen for anti-JNK1 (551197), Santa Cruz Biotechnology for anti-ubiquitin (P4D1), Bioword for anti-c-Jun (G237), Absci for anti-MKK4 (#AB21132) and anti-phospho-MKK4 (Ser80) (#AB11177), HangZhou HuaAn Biotechnology for anti-His2 at 37°C. Transfections were carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.HEK293T, HeLa, Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone) supplemented with 10% fetal bovine serum (FBS), 2.0 mM L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin. Cells were maintained in 5.0% COEscherichia coli (EHEC) O157:H7 Sakai strain (RIMD 0509952) and C. rodentium (CR) strain DBS100 (ATCC 51459) were used as wild-type strains. These bacteria were commonly cultured at 37°C LB broth. Deletion of nleL from EHEC O157:H7 genome was achieved through standard homologous recombination, as reported previously [nleL-deleted mutant and escR-deleted mutant had been constructed through a homologous recombination method “Gene doctoring” [nleL strain was transformed with plasmid encoding wild-type Flag-tagged NleL or its C753A mutant.Enterohemorrhagic eviously . For CR,ctoring” ,55. The 2 to induce the expression of type III secretion system before infection. For the complementation assay in EHEC or CR, the medium was added with 1.0 mM Isopropyl-B-D-thiogalactopyranoside (IPTG). Bacterial cells were then collected and suspended in PBS. After measuring the O.D. of cultured bacteria, infections were performed with mammalian cells at a multiplicity of infection (MOI) of 100:1 or 20:1, if not indicated otherwise, with a centrifugation at 800 g for 10 min, and then proceeded with incubation at 37°C in 5% CO2 for 2 ~ 3 h. After that, cells were washed three times and further cultured in fresh DMEM medium for another 2.5 ~ 5 h. Then the infected cells were subjected to immunofluorescence assay or IB analyses with indicated antibodies.The infection was performed as described before with slight modifications ,57. BrieE. coli BL21 (DE3) strains harboring the corresponding recombinant plasmids were grown in LB medium supplemented with appropriate antibiotics. Protein expression was induced overnight at 16°C with 0.3 mM IPTG when OD600 reached 0.6 ~ 0.8. To purify His6-tagged proteins, bacteria were harvested and lysed in lysis buffer containing 50mM Tris-HCl (pH 7.6), 300 mM NaCl, 40 mM imidazole and 5.0 mM beta-mercaptoethanol, and then proteins were purified with affinity chromatography using Ni-NTA beads (Qiagen) according to the manufacturer’s instruction. For GST-fusion proteins, purifications were performed by affinity chromatography using Glutathione Sepharose Fast Flow beads . Eluted proteins were further dialyzed overnight at 4°C. Recombinant proteins were concentrated and then frozen-stored in a buffer containing 50 mM Tris-HCl (pH 7.4), 300 mM NaCl and 15% Glycerol (V/V). Protein concentrations were determined using Bradford colorimetric assays, with their protein purities examined with SDS-PAGE followed by Coomassie Blue staining.Jnk1/2-specific shRNAs used were described before [Jnk1/2, lentiviral particles harboring specific shRNA expression vector  were produced by transfection of HEK293FT cells with the shRNA expression plasmid and lentiviral packaging mix. Target cells (HEK293T and HeLa) were incubated with the viral supernatant in the presence of 8 μg/ml polybrene (Sigma) and selected with 2 μg/ml puromycin (Clontech).Nucleotide sequences for the human d before . For stain vitro ubiquitylation assays were carried out in a 30 μl reaction system containing E1 (100 ng), His6-tagged UbcH7 (200 ng), GST-NleL or C753A (500 ng), Flag-JNK1 (500 ng) and ubiquitin (1.0 μg) in ubiquitylation buffer  at 37°C for 60 min. After the reaction was terminated by adding one-tenth volume of 10 × SDS sample buffer, the resulted mixtures were boiled and then subjected to SDS-PAGE, followed by IB analysis with indicated antibodies.As previously described with minor modifications , in vitrIn vivo ubiquitylation were also carried out as described previously [eviously . Cells wHEK293T cells in 6 cm dish were transfected with 5.0 μg pCDNA3-Flag-JNK1 and cultured for 24 h. Cells were then lysed in IP buffer , and centrifuged  for 15 min at 4°C. The supernatants were then subjected to IP with anti-Flag M2 beads to enrich Flag-JNK1. Then Flag-JNK1 was eluted by using 3 × Flag peptides (150 μg/ml in PBS) at 4°C for 30min. The eluted protein was verified by Coomassie Blue staining of SDS-PAGE gels.For Co-IP assay, 24 h after transfection with indicated plasmids, HEK293T cells were lysed in Triton X-100 buffer  plus 1% protease inhibitor cocktail at 4°C for 1 h. The corresponding antibody-conjugated Sepharose beads were added into the lysates supernatant. Following incubation overnight, the beads were washed five times (10 min each). For GST pull-down assay, purified GST-NleL, His-JNKs or cell lysate (appropriate volume) were mixed in total 400 μL reaction system  Triton X-100, 1 mM EDTA) at 4°C for 6 h. The beads were then pelleted and washed for 5 times (10 min incubation at 4°C for each washing). Then the recovered beads were boiled in 1× SDS-PAGE loading buffer and subjected to SDS-PAGE, followed by Coomassie Blue staining or IB with indicated antibodies.For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized for 10 min with 0.2% Triton X-100 in PBS, and then blocked for 60 min with 1.0% BSA, followed by incubation with indicated antibodies. All the cell nuclei were counterstained with DAPI before imaging.In vitro kinase assays were performed as described before [in vitro kinase reaction  containing 1.0 μg of GST-c-Jun (1-79aa) in the kinase buffer  supplemented with 2.0 mM ATP. The in vitro kinase assays involving human JNKs were carried out at 30°C for 60 min. Reactions were stopped by adding 1 × SDS-PAGE loading buffer and subjected to IB analysis with anti-p-c-Jun antibody.d before . Cells w2O, Promega) for 15 min at room temperature with vigorous shaking. AP-1 activities were finally determined using the dual luciferase assay kit (Promega). Results were independently replicated for at least three experiments.Luciferase reporter assays were performed as described previously . HEK293TGapdh) were assessed in triplicate wells of a 96-well reaction plate by subjecting 10 ng cDNAs per well to a Bio-Rad CFX96 Touch Detection System with SYBR Green chemistry using the following primers:Total RNAs from indicated cells were extracted using TRIzol (Invitrogen). RNA concentrations were determined on Nanodrop ND-1000 sepectrophotometer. 0.1 μg total RNAs were used to cDNA synthesis with the ReverTra Ace qPCR RT kit , according to the manufacturer’s instructions. The relative levels of genes expression  analysis as previously described . SEM samY2H screening with full-length NleL as the bait was performed as described previously .t-test. In all experiments, only P value of < 0.05 was considered to be statistically significant.Statistical significance of the data was determined using the Student’s S1 Fig(A) The growth of wild-type and ΔnleL EHEC O157:H7. Cultures grown to stationary phase were diluted 1:100 into fresh medium, and then the growth of each bacterium was monitored by measuring O.D. 600 at the indicated time points. (B) Several candidate genes were identified by using yeast two hybrid (Y2H) system. Full-length nleL was cloned into the bait vector pDEST32. Yeast two-hybrid screening was then performed with NleL as the bait, in a human ORFs library. The yeast clones which grow on SD-4 medium  were subjected to sequencing. (C) Human JNK1 interacted with NleL in vitro. GST-tagged wild type NleL (GST-NleL) or its C753A mutant (GST-NleL-CA) was co-immunoprecipitated with ectopically expressed JNK1. (D) GST pull-down assay of full-length NleL or its truncation mutants with JNK1. GST-tagged full-length NleL or its truncation mutants were individually mixed with lysates of the HEK293T cells expressing JNK1. After pull-down, Flag-tagged JNK1 was detected by IB analysis with anti-Flag. Blots are representative of at least three independent experiments.(TIF)Click here for additional data file.S2 FigA and B) NleL assembled poly-ubiquitin chains in vitro or in mammalian cells. In vitro ubiquitylation system assembled with purified GST-tagged NleL or C753A mutant, was incubated with E1, E2 (UbcH7), Ub at 37°C for 60 min (A). HEK293T cells were co-transfected with His6-tagged wild-type NleL or its C753A mutant and HA-tagged Ub. Poly-Ub chains or conjugates were determined by IB analysis with indicated antibodies (B). (C and D) NleL ubiquitylated JNK1 with preferred Ub chain linkages, especially K29-linked Chains. HEK293T cells were transfected with Flag-tagged JNK1 and His6-tagged NleL along with wild-type Ub or its mutants. Cell lysates were subjected to immunoprecipitation using anti-Flag M2 beads in denaturing RIPA buffer to enrich Flag-JNK1, followed by IB analysis with indicated antibodies. Blots are representative of at least three independent experiments.((TIF)Click here for additional data file.S3 Fig(A) Several E2s, especially UbcH7, are involved in NleL-mediated JNK1 ubiquitylation in vivo. Wild-type or specific-E2 knockdown HEK293T cells were co-transfected with plasmids encoding HA-tagged Ub, Flag-tagged JNK1, His6-tagged wild-type NleL. Flag-tagged JNK1 was immunoprecipitated with anti-Flag M2 beads in denaturing RIPA buffer, followed by IB analyses with anti-HA antibody. (B) NleL induced ubiquitylation of Flag-JNK1 or its mutants bearing Lys-to-Arg substitution at indicated sites in HEK293T cells. (C) The effect of JNK1 K68R mutant on NleL-induced poly-ubiquitylation of JNK1. All the blots are representative of at least three independent experiments.(TIF)Click here for additional data file.S4 FigResidues of 100%, over 80% or 60% homology in all aligned sequences are shaded in black, gray, or light gray, respectively. The residues responsible for JNK ubiquitylation by NleL were marked (red). Thr183 and Tyr185, the two conserved phosphorylation sites were labeled (blue color). The isoforms of human JNKs used in the alignment were JNK1α1, JNK2α2 and JNK3α2.(TIF)Click here for additional data file.S5 Fig(A) NleL promotes ubiquitylation of endogenous JNK2 in vivo. Cells expressing HA-tagged Ub and His6-tagged NleL (His-NleL) were lysed in 1.0% SDS buffer, boiled at 95°C for 10 min, and then diluted 10 fold in M2 buffer. Then HA-Ub-conjugated protein was enriched with anti-HA antibody and protein G beads, followed by IB analysis with anti-JNK2. (B) NleL promotes ubiquitylation of JNK2 with HA-tagged Lys 29 only Ub mutant. Cell lysates were subjected to immunoprecipitation using anti-Flag M2 beads and IB analysis with anti-JNK2 antibody. (C) Lys-to-Arg substitutions at Lys 68 and Lys 153 attenuated the ubiquitylation of JNK2. HEK293T cells were transfected with plasmids encoding His6-tagged NleL and Flag-tagged JNK2 or the mutant bearing Lys-to-Arg substitution at indicated sites . Cell lysates were subjected to IP with anti-Flag M2 beads in denaturing RIPA buffer, followed by IB analysis with indicated antibodies. (D) Wild-type NleL, but not the C753A mutant, inhibits the phosphorylation of ectopically expressed JNK2 in HEK293T cells. Blots are representative of at least three independent experiments.(TIF)Click here for additional data file.S6 Fig(A) NleL impaired the recruitment of endogenous MKK7 to JNK2. Lysates of HEK293T cells expressing Flag-tagged JNK2 together with or without His6-tagged NleL were subjected to anti-Flag IP. Then precipitated MKK7 was determined by IB with anti-MKK7. (B) NleL attenuated the interaction between ectopically expressed MKK7 and Flag-tagged JNK1 (left) or JNK2 (right). (C) NleL had little or no effect on MKK4 phosphorylation. HEK293T cells were stimulated by TNFα , and then IB analysis was performed with anti-p-MKK4 antibody. Blots are representative of at least three independent experiments.(TIF)Click here for additional data file.S7 Fig(A) NleL abolished TNFα-mediated JNK phosphorylation in the presence of PS-1145, an IKK inhibitor. H1299 cells expressing His6-tagged NleL or C753A were treated with TNFα (10 ng/ml) in the presence of PS-1145 (10 μM) for the indicated times. Phosphorylation status of the proteins was determined by IB with respective antibodies. (B) Ectopically expressed EGFP-NleL blocked c-Jun activation in HeLa cells with or without TNFα treatment. HeLa cells were stained with anti-p-c-Jun (red) and the nuclears stained with DAPI (blue). Immunofluorescence microscopy was performed. Scale bar, 10 μm. Representative images of at least three independent experiments are shown. The phosphorylation levels of c-Jun in EGFP-positive and EGFP-negative cells were further quantitated (down). Data are represented as the mean ± s.d. from at least four biological replicates, **P < 0.01, ***P < 0.001 . (C and D) NleL blocked the transcription activity of AP-1 induced by JNK1 overexpression (C) or PMA treatment (D). His-NleL and AP-1 reporter plasmids were transfected to HEK293T cells with (C) or without (D) Flag-tagged JNK1 for 24 h. Cells were stimulated by PMA  and then subjected to luciferase activity assay. Data are represented as the mean ± s.d., **P < 0.01, ***P < 0.001 . (E) NleL reduced 10% FBS-stimulated protein expression of cyclin D1 in starved cells. HEK293T cells expressing NleL or C753A mutant were serum-starved for 24 h and then stimulated with 10% FBS for indicated times. Then IB blottings were performed to determine protein level of cellular cyclin D1, respectively. (F) NleL suppressed thrombin-induced CCND1 expression. Cells expressing NleL or NleL-CA were serum-starved for 24 h, and then subjected to thrombin (1U/mL) treatment for at least 4 h. Blots are representative of at least three independent experiments.(TIF)Click here for additional data file.S8 FigA) C. rodentium strain DBS100 (ATCC 51459) was used as wild-type strain. The nleL-deleted mutant (ΔnleL) and escR-deleted mutant (ΔescR) had been constructed through a homologous recombination method “Gene doctoring”. Plasmids pTRC99A-NleL and pTRC99A-NleL-C753A are separately introduced to nleL-deleted mutant to generated NleL-complemented nleL-deleted strain (ΔnleL + pNleL) and C753A-complemented nleL-deleted strain (ΔnleL + pC753A). HeLa cells were infected with indicated C. rodentium strains with multiplicity of infection (MOI) of 100:1 for 2.5 h, washed with PBS and then re-cultured 2 h in fresh DMEM medium. Infected HeLa cells were thoroughly washed with PBS and then subjected to immunofluorescence microscopy analysis. Shown are representative cell images where anti-LPS staining indicates bacteria (red), DAPI staining marks the nucleus (blue). (B) Quantification of relative number of C. rodentium attached to cells in (A). Bars represent mean ± s.d. from at least five biological replicates, n.s., not significant .((TIF)Click here for additional data file.S9 FigA) HeLa cells were infected with indicated C. rodentium strains with multiplicity of infection (MOI) of 100:1 for 2.5 h, washed with PBS and then re-cultured 2 h in fresh DMEM medium. Infected HeLa cells were thoroughly washed with PBS and then subjected to immunofluorescence microscopy analysis. Shown are representative cell images where anti-LPS staining indicates bacteria (red), DAPI staining marks the nucleus (blue) and F-actin denotes the filamentous actin stained by Cyto-Painter Phalloidin-iFluor 488 Reagent (green). (B) 4~5 week-old C57BL/6 mice were intragastrically inoculated with 1 × 109 CFU C. rodentium strains. Viable stool bacterial counts, measured at 8 days after inoculation, are shown as mean ± s.e.m. of log10 colony-forming units (CFU) per gram faeces.((TIF)Click here for additional data file.S10 Fig(A) Quantification of EHEC O157:H7 attached to Caco-2 monolayer (grown for 6 days). Bars represent mean ± s.d. from at least five biological replicates, *P < 0.05, **P < 0.01, ***P < 0.001 . (B) NleL enhances the ability of EHEC O157:H7 to attach Caco-2 monolayer (grown for 21 days) by inhibiting JNKs. Caco-2 monolayers (grown for 21 days) treated with DMSO or JNK inhibitor SP600125 (10 μM) were infected with EHEC strains for 2.5 h, then washed with PBS and further cultured for 4 h in fresh medium. After infection, cells were subjected to immunofluorescence microscopy analyses.(TIF)Click here for additional data file.S11 Fig(A) Infection was performed with HeLa cells at a multiplicity of infection of 100:1. Two hours after infection, cells were washed and re-cultured with fresh DMEM for 5 h (replacing medium again at 2.5 h). In the SP600125 group, cells were treated with 5 μM SP600125 for 3 h before infection and 4 ~ 5 h in further culture after infection. Immunofluorescence microscopy was next performed. Representative cell images are shown from three independent experiments. (B and C) Quantification of actin pedestals (B) and EHEC O157:H7 attached to cells (C) in (A). Bars represent mean ± s.d. from at least five biological replicates, ***P < 0.001, n.s., not significant .(TIF)Click here for additional data file.


Using a transgenic C57Bl/6 mouse model of Cox-2 over-expression driven by the chicken lysozyme locus (cLys-Cox-2), which directs integration site-independent, copy number-dependent transgene expression restricted to macrophages, we demonstrated that stromal macrophage Cox-2 in colorectal  adenomas from cLys-Cox-2 x ApcMin/+ mice was associated with significantly increased tumour size (P = 0.025) and multiplicity (P = 0.025), compared with control ApcMin/+ mice. Transgenic macrophage Cox-2 expression was associated with increased dysplasia, epithelial cell Cox-2 expression and submucosal tumour invasion, as well as increased nuclear β-catenin translocation in dysplastic epithelial cells. In vitro studies confirmed that paracrine macrophage Cox-2 signalling drives catenin-related transcription in intestinal epithelial cells. Paracrine macrophage Cox-2 activity drives growth and progression of ApcMin/+ mouse colonic adenomas, linked to increased epithelial cell β-catenin dysregulation. Stromal cell (macrophage) gene regulation and signalling represent valid targets for chemoprevention of colorectal cancer.Genetic deletion or pharmacological inhibition of cyclooxygenase (COX)-2 abrogates intestinal adenoma development at early stages of colorectal carcinogenesis. COX-2 is localised to stromal cells (predominantly macrophages) in human and mouse intestinal adenomas. Therefore, we tested the hypothesis that paracrine Cox-2-mediated signalling from macrophages drives adenoma growth and progression  These pre-clinical studies led to investigation of the chemopreventative efficacy of selective COX-2 inhibitors in randomized, placebo-controlled colorectal polyp prevention trials in humans4. Predictably, both celecoxib and rofecoxib use were demonstrated to be associated with a significant reduction in polyp recurrence at colonoscopy, compatible with a future role for selective COX-2 inhibitors in colorectal cancer (CRC) chemoprevention7. However, long-term use of coxibs caused an excess of cardiovascular thrombotic events such as myocardial infarction8, limiting future use of this class of drugs for primary prevention of ‘sporadic’ CRC. Therefore, there is a requirement for novel approaches, which harness the clear anti-neoplastic efficacy of COX-2 inhibition, whilst minimizing or avoiding systemic toxicity.There is incontrovertible genetic and pharmacological evidence from rodent models that the inducible isoform of prostaglandin (PG) G/H synthase (PTGS2), also known as cyclooxygenase (COX), COX-2 plays an important role in the early stages of intestinal tumorigenesis during adenoma (or polyp) development15, rather than the dysplastic epithelial cells that eventually undergo malignant transformation. Several cell types are believed to contribute to the stromal COX-2-positive cell population in human and mouse intestinal adenomas including macrophages15, (myo)fibroblasts14 and endothelial cells12. In human tumours, the predominant COX-2-positive stromal cell population is the CD68-positive macrophage11.We, and others, have described that COX-2 is expressed predominantly by stromal cells in human and rodent intestinal adenomasin vitro16. More recently, others have provided evidence that paracrine COX-2-mediated signalling from stromal fibroblasts can drive proliferation of human CRC cells in vitro17.The predominant stromal cell localization of COX-2 in adenomas implies a role for paracrine Cox-2-mediated signalling during the early stages of intestinal tumorigenesis. We have demonstrated that mouse macrophage Cox-2 induces tumorigenic behaviour (including anchorage-independent growth and resistance to apoptosis) of non-transformed IEC-6 rat intestinal epithelial cells in vivo remains unclear. The ApcMin/+ mouse model of the early stages of intestinal tumorigenesis remains the predominant rodent model of ‘sporadic’ colorectal adenoma development for investigating chemoprevention strategies19. Despite the fact that the majority of adenomas are present in the small intestine (SI) and newer conditional mouse models, effectiveness of chemopreventive agents in the ApcMin/+ mouse model is highly predictive of clinical efficacy7. Therefore, we tested the hypothesis that increased expression of Cox-2 by macrophages promotes ApcMin/+ mouse intestinal tumorigenesis using a transgenic model of macrophage-specific Cox-2 over-expression.The role of paracrine COX-2-mediated signalling from stromal macrophages during the early stages of intestinal tumorigenesis lysozyme (cLys) gene locus directs copy number-dependent, integration site-independent, macrophage-specific transgene expression in mice22. We cloned a 5.8 kb mouse genomic Cox-2 clone consisting of all 10 exons and a 239 bp 3′-UTR fragment into the pIIIiLysVSal30 vector , was tested in stably transfected chicken HD11 macrophages. Inducible mouse Cox-2 expression was demonstrated in two independent stably transfected HD11 cell clones . Unlike G5 BMDMs, unstimulated BMDMs from the G25 line exhibited cLys-Cox-2 mRNA expression, which was induced further by (LPS) and γ-interferon (IFN), in parallel with endogenous mouse Cox-2 expression  and were bred to near homozygosity on the B6 background (N6 generation backcross) using an in-house real-time PCR method for transgene genotyping  repeats in large, transcriptionally silent intergenic regions25, and one of which was located in the centre of the first (360 kb) intron of the MAM domain containing glycosylphosphatidylinositol anchor 2 gene.The chicken tor Fig. 20. Functnes Fig. . The 18 PCR Fig. . Three fion Fig. . Therefoing Fig. . The hemDNA Fig. . Whole gcLys-Cox-2 mice demonstrated no observable phenotypic differences from wild-type (WT) B6 littermates and bred normally. As expected from a previous study of cLys transgene expression in mice23, transgenic Cox-2 protein was detected by immunohistochemistry in F4/80-positive splenocytes and hippocampal glial cells in cLys-Cox-2 mice  of Cox-2 in ApcMin/+ mouse tumour tissue compared with adjacent non-neoplastic colonic mucosa  although this was not evident in the largest colonic tumours, perhaps due to the small number of tumours greater than 6 mm in diameter . Consistent with accelerated colorectal tumorigenesis in transgenic animals, 5 of 10 (50%) colonic tumours from cLys-Cox-2 x ApcMin/+ mice were adenomas with severe epithelial dysplasia . In addition, 3 colonic tumours in cLys-Cox-2 x ApcMin/+ mice demonstrated evidence of localized deep invasion of the muscularis mucosae by nests of epithelial cells consistent with tumour progression towards adenocarcinoma . Subgroup analysis of adenomas restricted to proximal, middle or distal thirds of the SI similarly demonstrated no significant difference in stromal Cox-2 scores between transgenic and non-transgenic ApcMin/+ mouse adenomas (data not shown). There was also no evidence of adenomatous epithelial cell Cox-2 localisation in transgenic cLys-Cox-2 x ApcMin/+ mouse SI adenomas, unlike counterpart colonic tumours.By contrast, there was no increase in stromal Cox-2 immunoreactivity in transgenic cLys-Cox-2 x ApcMin/+ mice27. Therefore, we measured the CD31-positive MVD of transgenic and non-transgenic ApcMin/+ mouse colonic adenomas  indicative of increased angiogenesis, which could account for accelerated intestinal tumorigenesis in mas Fig. . Increasmas Fig. .Figure 5cLys-Cox-2 x ApcMin/+ mice. Castellone et al.28 have reported that PGE2 can stimulate β-catenin-related transcription in human CRC cells lacking Adenomatous Polyposis Coli function. It is also established that dysplastic epithelial cells in ApcMin/+ mouse adenomas exhibit increased levels and nuclear localization of β-catenin, following loss of the second Apc allele29. Therefore, we compared localization of β-catenin protein in transgenic and non-transgenic colonic tumours  compared with control cells  is increased in transgenic Cox-2-over-expressing ApcMin/+ mouse tumour progression in the colorectum, analogous to human colorectal polyp growth and malignant progression. This confirms stromal macrophage Cox-2 as a tumour-specific target for secondary chemoprevention of CRC. Previously, ‘knockout’ studies have implicated a role for Cox-2 in the early stages of intestinal tumorigenesis2. Despite one of the studies confirming that expression of Cox-2 in mouse mutant Apc intestinal adenomas was restricted to stromal rather than epithelial cells1, these reports stopped short of providing proof that stromal cell Cox-2 up-regulation could drive rodent intestinal tumorigenesis. Our data now provide a strong rationale for targeting COX-2 expression in tumour-associated macrophages as a CRC chemoprevention strategy that is unlikely to share the cardiovascular toxicity associated with systemic pharmacological COX-2 inhibition8. For example, tumour-associated macrophages are now recognised as a target for anti-cancer nanotherapy30. Stromal macrophages also secrete other pro-tumorigenic mediators including WNTs and multiple chemokines/cytokines32. However, it should be noted that other cell types contribute to stromal COX-2 expression in intestinal adenomas, at least in the mouse, namely fibroblasts and endothelial cells12.Our transgenic, macrophage-specific Cox-2 over-expression model provides the first direct evidence that increased stromal macrophage Cox-2 can drive ApcMin/+ mice was restricted to the colon and was not observed in the SI. This may be due to the larger stromal inflammatory infiltrate observed in large colonic tumours33, thus driving an increased transgenic Cox-2-dependent paracrine signal. Our data are compatible with those of Cherukuri and colleagues, who reported that myeloid cell-specific Cox-2 deletion in a Cox-2fl°x/fl°x x LysMCre/+ mouse model did not alter SI tumour number or size34. In that study, the small number of colonic tumours precluded specific analysis of the effect of myeloid cell-specific Cox-2 deletion in the colon34.Macrophage Cox-2-dependent tumour growth and progression in COX-2 up-regulation in intestinal adenomas. COX-2-positive tumour-associated macrophages in human (and mouse) intestinal adenomas exhibit a phenotype that is not entirely compatible with either classical (M1) macrophage activation or alternatively activated macrophages (M2). For example, Cox-2-positive tumour-associated macrophages in ApcMin/+ mouse adenomas do not express inducible nitric oxide synthase (Nos) 235, which would be expected in classically activated M1 macrophages37. Instead, F4/80-positive macrophages in ApcMin/+ mouse tumours express arginase I, a recognised M2 marker15. Moreover, COX-2-positive macrophages in human colorectal adenomas contain activated nuclear factor κB38, which has recently been implicated in maintenance of an M2 phenotype of tumour-associated macrophages39. Mosser and Edwards36 have proposed a classification of macrophage populations based on different activities , which can co-exist such that macrophages can exhibit features at either end of the macrophage phenotype spectrum with inherent plasticity. We hypothesize that Cox-2-positive, Nos2-negative, NFκB-positive macrophages in intestinal adenomas likely exhibit a ‘regulatory’ phenotype. Interestingly, pharmacological Cox-2 inhibition in the ApcMin/+ mouse has been reported to switch tumour-associated macrophages from an M2 to M1 phenotype33.Pharmacological inhibition of stromal macrophage COX-2 expression is dependent on understanding the mechanistic basis of macrophage infiltration and Cox-2 up-regulation in macrophages40. However, we have previously been unable to demonstrate an intestinal permeability defect in ApcMin/+ mouse intestine24. Intracellular CRC-associated E. coli has been demonstrated to drive persistent COX-2 expression in human THP-1 macrophages41. Alternative suggested stimulatory signals include mucins produced by neighbouring epithelial cells42, secondary bile acids43 and direct epithelial-to-stromal cytokine/chemokine signalling eg. by monocyte chemoattractant protein 144. Interplay with other immune cell types, including regulatory T cells, is also likely to modulate the macrophage phenotype45, although this has not been studied in detail in colorectal adenomas, as opposed to CRCs.The micro-environmental stimulus for macrophage Cox-2 up-regulation remains unknown. Predominant localization of Cox-2-positive cells below eroded surface epithelium in intestinal adenomas suggests that exposure to luminal antigen may explain ApcMin/+ mouse intestinal tumours15 and is predominantly localised to stromal cells in human colorectal adenomas ). PGE2 levels are quoted as pg PGE2/mg total protein.Intestinal tissue was homogenised in 1 ml ice-cold Lysis Reagent 1 (Biotrak prostaglandin (PG) E60 were cultured in Iscove’s-modified Dulbecco’s Medium with Glutamax™ supplemented with 8% (v/v) foetal calf serum, 2% (v/v) chicken serum, 100 U/ml penicillin, 100 U/ml streptomycin and 0.15 μM monothioglycerol . Stable transfection with cLys-Cox-2 and pMC1 Neo was performed by the calcium phosphate method in G418-containing selection medium (700 μg/ml).HD11 pro-macrophage cells35.Mouse bone marrow-derived macrophages were obtained and cultured as describedMacrophages were activated with either 5 μg/ml LPS alone for HD11 cells or 1 μg/ml LPS, in combination with 100 U/ml γ-IFN for BMDMs, for 16 hours.16.Rat IEC-6 intestinal epithelial cells were cultured in medium conditioned by γIFN/LPS-activated or non-activated RAW264.7 mouse macrophages, which had been cultured in the absence or presence of the selective COX-2 inhibitor SC-236, as described61 using a 1:5000 dilution of goat polyclonal anti-Cox-2 antibody (Santa Cruz sc-1745) and a 1:20000 dilution of mouse monoclonal anti-β-actin antibody  in Tris-buffered saline with 0.1% (v/v) Tween-20 and 1% (w/v) dried skimmed milk. Secondary antibodies were either peroxidase-conjugated (DAKO) for chemiluminescence with ECL Western Blot Substrate  or AlexaFluor 680- (Thermo Fisher Scientific) and IRDye800-conjugated  for measurement of Cox-2 protein levels, which were normalised to the β-actin protein level by near-infrared analysis using an Odyssey® detection system . Western blot analysis of rat IEC-6 β-catenin levels was performed using a 1:5000 dilution of mouse anti-chicken β-catenin antibody  as described62.Western blot analysis was performed as described61.Transfection of the β-catenin/T cell factor-reporter construct TOPflash and control construct FOPflash, followed by dual luciferase assay of reporter activity controlled for transfection efficiency, was performed as describedAll analyses were performed using SPSS version 20 and statistical significance was assigned if P < 0.05.Supplementary Information


BCR-ABL1-positive and, as recently shown, BCR-ABL1-like leukemias. The cell line Ba/F3 lentivirally transduced with mutant BCR-ABL1 constructs is widely used for in vitro sensitivity testing and response prediction to tyrosine kinase inhibitors. The transposon-based Sleeping Beauty system presented offers several advantages over lentiviral transduction including the absence of biosafety issues, faster generation of transgenic cell lines, and greater efficacy in introducing large gene constructs. Nevertheless, both methods can mediate multiple insertions in the genome. Here we show that multiple BCR-ABL1 insertions result in elevated IC50 levels for individual TKIs, thus overestimating the actual resistance of mutant subclones. We have therefore established flow-sorting-based fractionation of BCR-ABL1-transformed Ba/F3 cells facilitating efficient enrichment of cells carrying single-site insertions, as demonstrated by FISH-analysis. Fractions of unselected Ba/F3 cells not only showed a greater number of BCR-ABL1 hybridization signals, but also revealed higher IC50 values for the TKIs tested. The data presented highlight the need to carefully select transfected cells by flow-sorting, and to control the insertion numbers by FISH and real-time PCR to permit unbiased in vitro testing of drug resistance.Point mutations in the ABL1 kinase domain are an important mechanism of resistance to tyrosine kinase inhibitors (TKI) in  BCR-ABL1 are the genetic hallmarks of chronic myeloid leukemia (CML) and Ph-positive acute lymphoblastic leukemia (Ph+ ALL) [BCR-ABL1 fusion gene [BCR-ABL1-like leukemias involving deregulated tyrosine kinases [BCR-ABL1 fusion gene [in vitro sensitivity testing of novel TKIs and the prediction of resistance of emerging BCR-ABL1 mutants [BCR-ABL1 tyrosine kinase [BCR-ABL1 constructs which span over 5.8 kb. Moreover, lentiviruses tend to insert within transcriptionally active sites, thereby increasing the risk of mutagenesis and altered gene expression [50) values for individual TKIs reported for the same mutation in the BCR-ABL1 TKD might be attributable to this phenomenon [Sleeping Beauty (SB) system, a synthetic DNA transposon designed to introduce defined DNA sequences into the chromosomes of vertebrate animals and humans, provides an attractive alternative for genetic transformation and insertional mutagenesis  chromosome and the corresponding fusion gene ion gene , the allion gene . The intion gene , 5, and  kinases , 7. In C kinases -11. Clinion gene , 13. The mutants -19 is the kinase . This sye kinase . Neverthe kinase , thus ree kinase . This prpression -27. Insepression , can alsenomenon , 29. FinBCR-ABL1 constructs containing either wildtype (BCR-ABL1WT) or various mutants including G250E (BCR-ABL1G250E), E255V (BCR-ABL1E255V), T315I (BCR-ABL1T315I), F317L (BCR-ABL1F317L), and F359V (BCR-ABL1F359V). Lentivirally transduced Ba/F3 cell lines bearing the same BCR-ABL1 mutant constructs were tested for comparison. Fluorescence-activated cell sorting (FACS) with gating for low fluorescence was implemented to select cells displaying single gene construct insertions and homogenous transgene expression, and the efficacy of specific cell enrichment was documented by fluorescence in situ hybridization (FISH). The approach presented is applicable to cell line-based in vitro sensitivity testing of any mutation relevant in the clinical context, but was not designed for the monitoring of molecular remission or TKI resistance in patient samples. The data presented highlight the importance of using appropriately selected Ba/F3 cells for clinically relevant readout of TKI sensitivity testing in vitro.We have employed the SB system to establish Ba/F3 cell lines stably transfected with fluorescent proteins and BCR-ABL1WT, BCR-ABL1G250E, BCR-ABL1E255V, BCR-ABL1T315I, BCR-ABL1F317L/V or BCR-ABL1F359V constructs were generated either by SB- or by LV-mediated gene transfer. After selection of successfully transformed cells using a fluorescence protein marker and IL-3 depletion, cells were either sorted for low fluorescence, as displayed in Figure BCR-ABL1WT -certified reference laboratory. By contrast, unselected Ba/F3 cells  revealed variable and significantly higher BCR-ABL1 expression levels, apparently reflecting the predominant number of genomic insertions of the construct.The increased copy number of s Figure . Real-tis Figure . MoreoveBCR-ABL1 constructs introduced either by SB-assisted transfection or LV-mediated transduction were subjected to in vitro survival assays with increasing concentrations of nilotinib, dasatinib, and ponatinib. The IC50 values obtained are displayed in Table 50 values corresponding to the lowest measurements published previously [BCR-ABL1T315I LV-transduced Ba/F3 cells which displayed multiple insertions of the construct, as documented by FISH analysis  according to the manufacturer's instructions. Puromycin (Sigma-Aldrich) was added at 1 μg/ml to the medium 24 h after transfection, followed by selection for 2-5 days. All cells positive for the fluorescent protein tdTomato were isolated by flow-sorting using the FACSAriaFusion instrument  and grown in IL-3-containing medium to a density of 0.8-1 × 106 cells /ml. IL-3-independent growth was assessed by washing the cells three times with IL-3-free medium followed by expansion in IL-3-free medium to 0.8-1 × 106/ml. Finally, the cells were either left unsorted or enriched according to a defined low-fluorescence range  where the cells are authenticated using PCR-based methods. Ba/F3 were maintained in RPMI 1640 medium  containing 10% fetal bovine serum (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific) and supplemented with 2 ng/ml murine IL-3 . Cells were seeded at 3 × 10e Figure , and useF3 cells  employedBCR-ABL1 construct insertions in the genome. Sheep antidigoxigenin-fluorescein isothiocyanate 1:100  was used for immunodetection of hybridized probes. Signals were analyzed using a ZEISS Imager M2 fluorescence microscope  and the Genesis software .The analysis by FISH was performed essentially as described . BrieflyBCR-ABL1-expressing Ba/F3 cells were seeded in 96-well plates at 3 × 105 cells/ml and incubated with the respective TKI for 72 h at 37°C/5% CO2. Survival was assessed using the Vybrant® MTT Cell Proliferation Assay Kit (Thermo Fisher Scientific) according to a protocol provided by the manufacturer. To estimate the IC50 of individual drugs for wildtype and mutant cells, smoothed dose-response curves were fitted using OriginPro 6.1 software . The IC50 values were calculated by determining the mean of three independent experiments each performed in quadruplicates.The TKIs nilotinib, dasatinib and ponatinib  were obtained as 10 mM stocks in DMSO. Imatinib was not included because of its restricted efficacy in the presence of mutations. Serial TKI dilutions were prepared in phenol red-free RPMI 1640 containing 10% fetal bovine serum, and 1% penicillin-streptomycin, (Thermo Fisher Scientific) for subsequent in vitro sensitivity assays. 6 Ba/F3 cells SB-transfected or LV-transduced with BCR-ABL1 were lysed using high-salt buffer  with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein content was assessed using the Bio-Rad Protein Assay Kit II . After denaturing the samples in NuPAGE® LDS Sample Buffer (Thermo Fisher Scientific), proteins were separated by electrophoresis using NuPAGE® 4-12% Bis-Tris Protein Gels (Thermo Fisher Scientific), transferred onto PVDF membranes (Thermo Fisher Scientific) with the XCell II™ Blot Module (Thermo Fisher Scientific), and treated with Odyssey Blocking Buffer TBS . Antibodies directed against the following targets were used to probe the membranes: phospho-Akt (Ser473), phospho-Gab2 (Tyr452), phospho-Crkl (Tyr207), Akt, Gab2, Crkl  and GAPDH . The DyLight™ conjugated antibodies  were used for visualization of specific bands.A total of 15×10BCR-ABL1 double- or single-stranded DNA molecules was quantified on the Taqman 7500 Real Time PCR System (Thermo Fisher Scientific) using the primer probe set Hs03024784_ft (Thermo Fisher Scientific). Taqman mouse copy number reference assays Trfc and Tert (Thermo Fisher Scientific) were used for normalization of the number of BCR-ABL1 inserts into the murine genome. The primer/probe set Mm01197698_m1 recognizing murine Gusb transcripts was used for normalization of the number of BCR-ABL1 transcripts. All experiments were done twice in triplicates. The standard curves were built using the plasmids pITR-ETP-BCR-ABL1WT, pCMV6-Gusb (Origene) and mouse genomic DNA (Promega). Relative expression levels and copy number variations were calculated according to the ΔΔCt method.Genomic DNA was isolated using the DNeasy Blood and Tissue Kit (QIAGEN). Total RNA was isolated with the help of RNeasy Plus Mini Kit (QIAGEN) and reverse-transcribed by employing the High Capacity cDNA Synthesis Kit (Thermo Fisher Scientific). The number of In the experiments described, two independent samples were analyzed in triplicates, unless indicated otherwise. Statistical analysis was performed using the OriginPro 6.1 software (OriginLab), and the two-sample independent T-test was employed for the calculation of significance values.


Although extracellular nucleotide, adenosine 5′-triphosphate (ATP), is also known as most potent Ca2+-mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca2+ signaling requires inositol 1,4,5-trisphosphate (IP3) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca2+/NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP3-mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca The sus pathway .3-mediated Ca2+ release from Ca2+ store  proteins are now accepted as molecular entities of receptor-activated cation channels . There a2+ store . Studiesertrophy . Inhibitertrophy .2+ signaling pathway to initiate various biological processes. Although ATP activates Gq- PLC-dependent Ca2+ signaling pathway in cardiac cells , has long been recognized as an endogenous ligand to stimulate purinergic signaling . Evidencac cells , it has ac cells .3-dependent Ca2+ signaling and NO production in neonatal rat cardiomyocytes (NRCMs), as well as functional coupling between IP3-responsive TRPC5 channel and endothelial NOS (eNOS) contributes to negative regulation of hypertrophic signaling induced by ATP stimulation.Paracrine modulation of cardiac excitation-contraction coupling (ECC) has recently become a topic of intense interest. Resident heart endothelial cells are well-known physiological paracrine modulators of cardiac myocyte ECC mainly via nitric oxide (NO) and ATP . We prevN-acetyl-DL-penicillamine (SNAP), 1-oleoyl-2-acetyl-sn-glycerol (OAG), ET-1, U73122, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) and 2-Aminoethyl diphenylborinate (2-APB) were from Sigma. Ang II was from Peptide Lab. AR-C 118925XX, UTPγS trisodium salt, SCH58261 and MRS1754 were from Tocris Bioscience. Fura 2-AM and Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) were from Dojindo. Diaminofluorescein-2 diacetate (DAF-2 DA) was from Molecular Probes. Dulbecco’s modified Eagle’s medium (DMEM) was from Wako. Anti-TRPC5, anti-TRPC6 antibodies were from Alomone. Anti-phospho-eNOS (Ser1177), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were from Santa Cruz Biotechnology. Anti-eNOS antibody was from BD Biosciences. Plasmids encoding NFAT promoter-dependent luciferase and brain natriuretic peptide (BNP) promoter-dependent luciferase, and dual luciferase reporter assay system were from Promega. Collagenase II was from Worthington. Stealth siRNAs for rat TRPC5 and TRPC6, Lipofectamine 2000, and Lipofectamine RNAiMAX reagent were purchased from Invitrogen.4-{amino}-6-methoxyquinazoline (MQ), KT5823, ionomycin and xestospongin C were purchased from Calbiochem. 8-bromo- cyclic guanosine 3′,5′-monophosphate (8-Br-cGMP), S-nitroso-2, 95% air) for 1 h in DMEM containing 10% FBS and 1% penicillin and streptomycin. Floating cells were collected and plated into gelatin-coated culture dishes at a density of around 1.5 × 105 cells/cm2. After 24 h, the culture medium was changed to serum-free DMEM. We confirmed that >90% attached cells were actinin-positive by immunostaining. For TRPC knockdown, NRCMs were transfected with each siRNA (50 nM) using Lipofectamine RNAiMAX for 72 h . DAF-2 DA was excited by 470 ± 20 nm, and the fluorescence images at the wavelength of 525 ± 20 nm were acquired using fluorescence microscopy . The fluorescence intensity of the digital images were analyzed using Image J software. Fold increases in fluorescence were calculated by subtracting fluorescence intensity before stimulation from that after stimulation, which was subsequently divided by that of no-stimulation.Measurement of intracellular Caescribed . After aMeasurement of NFAT-dependent luciferase activity and BNP promoter-dependent luciferase activity was performed as described previously , 2011.6 cells) plated on 6-well dishes were directly harvested with 2 × SDS sample buffer (200 μl). After centrifugation, supernatants (20–40 μl) were fractionated by 8% SDS-polyacrylamide gel and then transferred onto polyvinylidene difluoride membrane. The expression and phosphorylation of endogenous TRPC proteins were detected by anti-TRPC6  and anti-TRPC5 (1:1000) antibodies. We visualized the reactive bands using Western Lightning Plus ECL (PerkinElmer). The optical density of the film was scanned and measured with Scion Image software.NRCMs (1 × 103H]leucine incorporation (3H]leucine (1 μCi/ml) was added to the culture medium and further incubated for 4 h. The incorporated [3H]leucine was measured using a liquid scintillation counter.Measurement of hypertrophic responses was performed by measuring the transcriptional activation of BNP gene as described previously . Proteinporation . After ct-test (for two groups) or analysis of variance followed by Tukey’s post hoc test (for multiple groups). Values of P < 0.05 were considered significant. We made utmost effort to minimize the replicates of animal experiments according to the ethical guideline of 3R .Results are presented as the mean ± SEM. Statistical comparisons were made using Student’s 2+-NFAT signaling in NRCMs, as well as known hypertrophy-inducible ligands, ET-1 . ATP increased NFAT-dependent transcriptional activity more potently than ET-1 and Ang II . In contrast, despite increasing NFAT activity, ATP never increased hypertrophic responses, including protein synthesis determined by [3H]leucine incorporation and transcriptional activation of BNP . These results indicate that ATP-induced increases in [Ca2+]i and NFAT activity are not sufficient to induce hypertrophic responses in NRCMs.We first investigated the effect of ATP on Cads, ET-1  and Ang ds, ET-1 . ATP sho2 receptor stimulation , an inhibitor of NOS, resulted in significant enhancement of ATP-induced sustained NFAT activation to the same extent of that by ET-1 stimulation, while L-NAME had no impact on ET-1-induced NFAT activation. L-NAME also enhanced BNP transcriptional activation in response to ATP stimulation . The enhanced BNP activity was canceled by the treatment with MQ (10 μM), a phosphodiesterase (PDE) 5 selective inhibitor, suggesting the involvement of cGMP-dependent pathway. ATP produced a small transient increase in [Ca2+]i in the absence of extracellular Ca2+ , which was mainly derived from intracellular IP3-responsive Ca2+ store. Replenishing extracellular Ca2+ led to sustained increases in [Ca2+]i, which was derived from Ca2+ influx probably through store-operated Ca2+ channels . Treatment with L-NAME, but not D-NAME , significantly suppressed sustained [Ca2+]i increase . In contrast, although ET-1 stimulation also induced sustained [Ca2+]i increase, L-NAME failed to suppress the ET-1-induced sustained [Ca2+]i increase in NRCMs . These results suggest that ATP induces NO production in NRCMs and negatively regulates Ca2+/NFAT-dependent hypertrophic signaling.We next examined which signal pathway(s) induced by ATP stimulation negatively regulates hypertrophic growth of NRCMs. Previously we reported that mechanical stretch on NRCMs induces ATP release, which leads to NO production through P2Ymulation . As NO hmulation , we inveFigures 3A,B). Heart expresses all three isoforms of NOS . We further measured the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a substrate of PKG . These results indicate that ATP induces activation of NO-cGMP-PKG pathway in NRCMs.We next examined whether ATP actually induces activation of NO-dependent signaling in NRCMs. Compared to stimulation of NRCMs with ET-1 and Ang II, stimulation with ATP significantly increased fluorescence intensity of DAF-2, an NO-sensitive dye , while significantly reducing ET-1-induced increases in [Ca2+]i and NFAT activity . The ATP-induced Ca2+ response was completely suppressed by the treatment with xestospongin C (20 μM), an IP3 receptor (IP3R) blocker . As eNOS activity is predominantly regulated by the increase in [Ca2+]i as well as its phosphorylation, we further investigated whether IP3-dependent store-operated Ca2+ influx signaling mediates eNOS activation by ATP stimulation. Store-operated Ca2+ influx was evoked by the treatment with ionomycin (1 μM), a Ca2+ ionophore . The ionomycin-induced NO production was completely diminished in the absence of extracellular Ca2+, indicating that store-operated Ca2+ influx is required for ATP-induced NO production in NRCMs. Additionally, inhibition of IP3R or PLC suppressed ATP-induced NO production in NRCMs . These results suggest that ATP-induced Ca2+/NFAT signaling and NO production are not mediated by DAG-activated channels, including TRPC6.We previously reported that TRPC6, rather than TRPC3, predominantly mediates mechanical stretch-induced global i and NFAT activity were not reduced by TRPC5 knockdown . We also found that TRPC5 knockdown markedly reduced ATP-induced NO production , and increased ATP-induced BNP transcriptional activity at a rate similar to ET-1 stimulation . The induction of hypertrophic response by ATP stimulation in TRPC5 knockdown NRCMs were mimicked by the treatment with KT5823 . These results suggest that TRPC5 acts as negative regulator of hypertrophic signaling in NRCMs through eNOS-mediated activation of NO/cGMP/PKG signaling.TRPC5 has been shown to be upregulated in human failing heart, although its physiological role is still not fully understood . TRPC5 ial cells . We thus2R by AR-C 118925XX (10 μM) . Stimulation with UTPγS, a P2Y2/4R-selective ligand, also increased NO production. In addition, U73122 suppressed the ATP-induced Ca2+ response . As AR-C 118925XX has yellow-colored self-fluorescence that entirely overlaps F340 intensity of Fura-2, we could not measure the exact ratiometric changes of fura-2 induced by ATP stimulation in the presence of AR-C118925XX. However, we confirmed that AR-C 118925XX completely suppressed the ATP-induced increase in F340 intensity of Fura-2 (data not shown). These results strongly suggest that P2Y2R-PLC axis predominantly mediates ATP-induced Ca2+ response and NO production in NRCMs.We finally examined which purinergic receptor subtype(s) mediates ATP-induced NO production in NRCMs. As expected, the ATP-induced NO production was significantly suppressed by pharmacological inhibition of PLC by U73122 (1 μM) or P2Y1R-selective antagonist), SCH58261 , and MRS1754  did not significantly suppress ATP-induced NO production . Therefore, adenosine receptors may hardly contribute to ATP-induced anti-hypertrophic NO signaling in NRCMs.Extracellular ATP is rapidly converted into ADP, AMP and adenosine by membrane-bound ectonucleotidases . Adenosi2+ also suppressed ATP-induced Ca2+ response and NO production, some Ca2+ influx pathway(s) may be activated by ATP stimulation. However, the context of store-operated Ca2+ channels (SOCs) are hardly developed in matured cardiomyocytes, and the treatment with 2-APB (10 μM), an inhibitor of SOCs, suppressed Ca2+ response but not NO production induced by ATP stimulation. Thus, IP3-dependent TRPC5-mediated Ca2+ influx pathway, but not other Ca2+ release-activated Ca2+ influx pathway, is involved in ATP-induced Ca2+-dependent NO production in NRCMs.As the removal of extracellular Ca2+/NFAT signaling in cardiac hypertrophy is well established, and local Ca2+ influx through TRPC3/6 channels may be a putative mechanism underlying activation of calcineurin-NFAT signaling pathway in rodent myocardium. We demonstrated that ATP-induced increases in [Ca2+]i and NFAT activity are not sufficient to induce hypertrophic responses in NRCMs. Previous studies revealed that sustained Ca2+ oscillation is more efficient in activating NFAT-dependent hypertrophic gene expression than transient Ca2+ rise induced by hypertrophy-inducible ligands and mechanical stretch  .Although the physiological importance of NO-dependent signaling in heart has been long discussed, the major origin of NO was generally thought to be endothelial cells because of poor expression levels of NOS enzymes in NRCMs. However, local activation of NO signaling in cardiomyocyte induced by sex hormone receptor stimulation has been also attracting attention as a negative regulatory mechanism of cardiac arrhythmia . The rol stretch . We reveertrophy . We founin NRCMs , 2011. WS (iNOS) . In this2+ influx negatively regulates ATP-induced hypertrophic response of NRCMs through activation of NO signaling. Indeed, suppression of TRPC5 resulted in reduction of ATP-induced NFAT activation and NO production, thus promotes hypertrophic response in NRCMs. Although NFAT activation has been long associated with hypertrophic gene expression, we speculate that the transient activation of NFAT mediated by TRPC5-dependent Ca2+ entry is not sufficient to induce hypertrophic gene expression in NRCMs.Although TRPC5 expression is upregulated in pathologic hypertrophied human hearts , its phy2R stimulation in neonatal cardiomyocytes is also applicable to adult cardiomyocytes. As TRPC5-mediated NO signaling requires IP3-mediated Ca2+ release and IP3-mediated Ca2+ signaling is down-regulated in adult cardiomyocytes compared to that in neonatal cardiomyocytes, contribution of TRPC5-eNOS axis might be minor in normal adult cardiomyocytes. Future study using adult cardiomyocytes will be necessary to elucidate the pathophysiological role of TRPC5-eNOS axis in heart.Because of limitation of the study, we could not determine whether the TRPC5-eNOS axis induced by P2Y2+/NFAT-dependent hypertrophic response of NRCMs. Purinergic receptors are well accepted as an attractive therapeutic target of age-related cardiovascular diseases (2R agonists in the prevention of cardiac hypertrophy. Our new finding will provide a new therapeutic strategy for the prevention of pathological cardiac hypertrophy.In summary, we revealed a physiological role of TRPC5 channel in rat cardiomyocytes. TRPC5 functionally couples with eNOS, and activation of TRPC5-mediated NO signaling induced by ATP stimulation negatively regulates Cadiseases , and we MN supervised and conceived the project. CS, KS, and SO designed the experiments and prepared the manuscript. CS, KS, SO, TT, KN, and SM performed the experiments and interpreted the data. MN, AN, and TN-T edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AA-T and handling Editor declared their shared affiliation.