IFNB1 and CXCL10. Taken together, this study demonstrates the existence of a post-translational regulation of STING by HCMV IE86 protein.Stimulator of interferon genes (STING) is a critical signaling molecule in the innate immune response against DNA viruses by either directly sensing intracellular DNA or functioning as an adaptor molecule to activate the type I interferon (IFN) signaling pathway. We determined the functional interaction between STING and human cytomegalovirus (HCMV). A cDNA library containing 133 HCMV ORFs was screened to identify viral genes that inhibit STING-induced IFN-β promoter activation. Among the screened ORFs, UL122, which encodes the immediate-early 2 86 kDa (IE86) protein, strongly abolished STING-induced IFN-β promoter activation. Interestingly, IE86 protein facilitated the proteasome-dependent degradation of STING and inhibited 2′3′-cGAMP-mediated induction of  Stimulator of interferon genes (STING), also known as TMEM173, ERIS, MITA and MPYS, is a critical signaling molecule that plays a key role in the type I IFN signaling pathways in response to DNA virus infection  through the Golgi to the perinuclear location and post-translational modifications such as phosphorylation or ubiquitination  and monocytic leukemia cell line THP-1 . DisruptAlthough STING plays a pivotal role in DNA virus infection-induced innate immune responses, a regulatory mechanism(s) for STING employed by HCMV protein(s) has not been extensively elucidated. Other viruses have been reported to employ effective mechanisms to counteract the STING pathway  protein most effectively reduced STING-induced IFN-β promoter activation. Potent inhibition of STING-induced IFN-β promoter activity by IE86 protein was expected because IE86 protein inhibits HCMV-induced IFN-β production by interfering with NF-κB binding activity to the IFN-β promoter , 2006. ITM lentiviral expression system and retroviral gene transfer and expression system, respectively, according to the manufacturer’s directions (Clontech). The propagation of HCMV–Towne was described previously , IE86 (Ad-IE86), green fluorescent protein (GFP) (Ad-GFP) or tetracycline transactivator (Ad-Trans) were described previously (The maintenance and propagation of primary human foreskin fibroblast (HFF) and HEK293T cells were described previously . Recombieviously . Standareviously . Propagaeviously . The traeviously .TM for transient transfection was used according to the manufacturer’s directions (Omics Biotechnology). The luciferase assay was performed as described previously pA(3′5′)p] (2′3′-cGAMP) was purchased from InvivoGen. Omicsfecteviously .TM enzyme mix (Invitrogen). National center for biotechnology information (NCBI) reference sequence number for hSTING used in this study is NM_198282.1. 6X-myc-IE86 wild type (WT) or deletion mutants were cloned into pCS3-MT (with a 6X-myc tag) -based destination vector from the pENTR-IE86 WT or deletion mutants using LR clonaseTM enzyme mix (Invitrogen) as described previously (TM enzyme mix (Invitrogen). pEF-Bos TRIF-FLAG was a gift from Kate Fitzgerald and Tom Maniatis (Addgene plasmid # 41550) (The shRNA sequences for human STING (hSTING) to generate pLKO.1-STING shRNA-a and -b were obtained from the genetic perturbation platform (GPP) at Broad Institute . pLKO.1 scramble shRNA was a gift from David Sabatini (Addgene plasmid #1864) . To geneeviously . IE72, I# 41550) .TM enzyme mix according to the manufacturer’s directions (Invitrogen).A HCMV-Towne ORF library in the pENTR vector (Invitrogen) was described previously . One huno-nitrophenyl-β–galactopyranoside (ONPG) assays. Yeast strains, media for yeast growth, methods for yeast transformation, and β-galactosidase assays were all as described previously (Yeast AH109 (MATa) cells were transformed with plasmid (Trp+) expressing the GAL4-DNA-binding domain (DBD)-STING fusion protein. Y187 (MATa) cells were transformed with plasmid (Leu+) expressing the GAL4-activation domain (AD)-HCMV ORF fusion proteins. Transformants were selected on plates lacking tryptophan or leucine. Trp+ and Leu+ transformants were mated with each other on complete YPD plates and diploid cells (a/α) were selected on plates lacking both tryptophan and leucine. Cells expressing bait and prey that interact with each other grow on plates that lack tryptophan, leucine, and histidine and express β-galactosidase. Cells expressing GAL4-DBD-STING and GAL4-AD only were used as a negative control. For rapid in site assays for β-galactosidase production, a 5-bromo-4-chloro-3-indolyl-β–galactopyranoside  filter assay was used. For quantitative assays, β-galactosidase production was measured using eviously .TM software (Bio-Rad Laboratories).Cells were harvested, fractionated and transferred onto nitrocellulose membranes as described previously . AntibodThe isolation of total DNA and analysis of HCMV replication using quantitative PCR (qPCR) were performed as described previously . The isoFigure 1A). Compared with pLKO.1-scramble shRNA-transduced cells, the viral titer was increased 4.3-fold and 2.9-fold in cells transduced with pLKO.1-STING shRNA-a and -b, respectively . Since STING KD was more efficient with pLKO.1-STING shRNA-a than -b, HFF cells stably transduced with pLKO.1-STING shRNA-a were used for the following experiments. In STING KD HFF cells, HCMV infection-induced expression of IFNB1 transcripts was significantly reduced by 2.8-fold compared with control cells . The residual expression of IFNB1 transcripts induced by HCMV infection in STING KD cells was possibly mediated by the remaining STING protein and/or other innate immune signaling pathways such as Toll-like receptor (TLR) 2 and 9  (HFF cells stably transduced with either pLKO.1-scramble or two different pLKO.1-STING shRNAs (a and b) were infected with HCMV-Towne, and, at 7 days after infection, the viral titer (pfu/ml) was determined using plaque assays. In cells stably transduced with pLKO.1-STING shRNA-a and -b, the level of STING protein was significantly reduced by 78 and 61%, respectively ( with 1) .Figure 1C).To quantitatively analyze the level of HCMV replication in HFF cells stably transduced with pLKO.1-STING shRNA, cells were infected with the HCMV-Towne at low (0.1 pfu/cell) or high (1 pfu/cell) multiplicity of infection (MOI), and the relative amount of viral DNA was measured by qPCR using primers specific for UL123 at 5 days after infection. In STING KD HFF cells, HCMV replication was significantly increased by 2.9-fold at low MOI and 3.4-fold at high MOI compared with control cells . These data indicate that STING KD up-regulates the expression of HCMV lytic genes and induces HCMV replication.The levels of UL122 (IE), UL44  or UL83  transcripts were also determined by qRT-PCR. STING KD induced the mRNA levels of HCMV IE, E and L genes by 2-, 8-, and 10-folds, respectively, at low MOI or by 3.6-, 5.6- and 2.3-folds, respectively, at high MOI . Ectopic expression of STING protein induced the level of IFNB1 transcripts 3.3-fold . In HFF cells transduced with pLHCX-STING-myc retroviral vector, HCMV infection-induced IFNB1 expression was further induced 2.1-fold compared with control cells . To quantitatively analyze the level of HCMV replication in HFF cells stably transduced with either pLHCX- or pLHCX-STING-myc retroviral vector, the relative amount of viral DNA was measured by qPCR using primers specific for UL123. In HFF cells over-expressing STING, HCMV replication was significantly reduced by 86.7% at low MOI (0.1 pfu/cell) and 71.9% at high MOI (1 pfu/cell) compared with control cells . Taken together, these data suggest that STING plays an important role in limiting HCMV replication.To further determine the effect of STING on HCMV infection, HFF cells stably transduced with either pLHCX- or pLHCX-STING-myc retroviral vector were generated  in cells co-transfected with the vector expressing STING and a vector expressing HCMV-Towne ORF was divided by that in cells co-transfected with control vector and a vector expressing HCMV-Towne ORF. To analyze the relative luciferase activity, STING-induced luciferase activities without HCMV-Towne ORF were set to 100%. Among the screened HCMV-Towne ORFs, UL25, UL36, UL82, UL89B, UL94, UL122 and US23 reduced STING-induced IFN-β promoter activation by greater than or equal to 50% . HCMV UL122 encoding IE86 protein most effectively reduced STING-induced IFN-β promoter activation by 87% .To identify HCMV genes that interfere with STING-induced type I IFN response, a cDNA library containing 133 HCMV-Towne ORFs was screened using IFN-β promoter-driven luciferase reporter. HEK293T cells, which do not express detectable STING protein , were coFigure 4) . Among the screened HCMV-Towne ORFs, UL122 exhibited the strongest interaction with STING . However, Gal-4 AD fusion with HCMV UL25, UL36, UL82, UL89B, UL94 and US23, that reduced STING-induced IFN-β promoter activation, was less sensitive for the detection of β-galactosidase. In addition to HCMV UL122, Gal-4 DBD fused to STING interacted with a Gal-4 AD fusion with HCMV UL78, UL100, UL132, UL148A, UL148B, US14 and US18 .The interaction between STING and HCMV-Towne ORFs was also investigated by a yeast two-hybrid assay as previously described (igure 4) . InteresFigure 5A, compare lane 2 with 1). Since IE86 protein inhibits STING-induced type I IFN promoter activation, the effect of IE86 protein on STING expression was determined. HFF cells were transduced with 10 pfu per cell of Ad-IE72, Ad-IE86 or Ad-GFP. At 48 h after transduction, the level of STING protein was determined by western blot analysis. Interestingly, the protein level of STING, but not TBK1, was significantly reduced by 4.8-fold in Ad-IE86-transduced cells compared to Ad-GFP-transduced cells . The mRNA level of STING was not attenuated in cells transduced with Ad-IE86 . Compared to IE86 protein, IE72 protein had no effect on the level of STING protein .As previously reported , HCMV inFigure 6A). Both MG132 or epoxomicin treatment restored the level of STING protein in Ad-IE86-transduced cells similar to that in Ad-GFP-transduced cells, indicating that IE86 induces proteasome-dependent degradation of STING protein . Although microtubule-associated protein light chain 3 (LC3) isoform B (LC3B) was significantly accumulated in cells treated with chloroquine, the level of STING protein was restored to a lesser extent than in cells treated with proteasome inhibitors . Thus, IE86 protein induces STING degradation mainly via a proteasome-dependent pathway.To determine whether IE86 protein induces STING degradation via a proteasome- or lysosome-dependent pathway, cells were treated with either proteasome inhibitors, MG132 and epoxomicin, or a lysosome inhibitor, choloroquine . Cells transduced with either Ad-GFP or Ad-IE86 were pretreated with MG132 for 12 h and followed by a cycloheximide chase for the indicated time points. In Ad-GFP-transduced cells, the endogenous STING protein half-life was approximately 50 min. On the other hand, the half-life of STING protein was substantially reduced (approximately < 25 min) in cells transduced with Ad-IE86 . The half-life of TBK1 protein was reported to be approximately 2.5 h in NIH3T3 cells . These data further support the hypothesis that IE86 protein promotes the proteasome-dependent degradation of STING protein.Since IE86 protein reduced the steady-state protein level of STING, the effect of IE86 protein on the half-life of STING protein was determined using a pulse-chase experiment . Since HEK293T cells do not express detectable STING . Ectopic expression of STING in these transfection settings enhanced IFN-β promoter activity 4-fold, and 2′3′-cGAMP treatment further activated to 9.4-fold . In HEK293T cells expressing ectopic STING protein, both IE86 and UL82 proteins suppressed 2′3′-cGAMP-induced IFN-β promoter activity by 87 and 56%, respectively . The IE72 protein had no effect on 2′3′-cGAMP-induced IFN-β promoter activation . Both IE86 and UL82, but not IE72, proteins significantly reduced the level of STING protein .To determine whether HCMV IE86 protein interferes with STING-induced type I IFN pathway, HEK293T cells were co-transfected with small amounts of vectors expressing STING and control  pathway . At 12 hle STING , 2′3′-cGIFNB1 and CXCL10 were determined using qRT-PCR at 12 h after treatment. In cells transduced with Ad-GFP, 2′3′-cGAMP treatment induced levels of IFNB1 and CXCL10 2.5- and 610-folds, respectively . The expression of IE86 protein significantly reduced 2′3′-cGAMP-induced levels of IFNB1 and CXCL10 by 56 and 67%, respectively . Thus, these results suggest that HCMV IE86 protein blocks STING-induced signaling pathway.To further determine the effect of IE86 protein on 2′3′-cGAMP-induced ISG expression, HFF cells transduced with Ad-GFP or Ad-IE86 were treated with 2′3′-cGAMP, and levels of ISGs such as Figure 8A). In HEK293T cells, overexpression of STING or TRIF protein induced phosphorylation of TBK1 at serine 172 . IE86 protein had no effect on both the level of TRIF protein and TRIF-induced TBK1 activation . However, the level of STING protein and STING-induced TBK1 activation were significantly reduced in cells expressing IE86 protein .To ascertain the effect of IE86 protein-mediated degradation of STING protein on STING-induced signaling pathway, HEK293T cells were transfected with the vector expressing STING or Toll/IL-1 receptor (TIR)-domain-containing adaptor-inducing IFN-β (TRIF), and TBK1 phosphorylation at serine 172, indicative of TBK1 activation, was determined . Ectopic expression of STING and TRIF strongly activated IFN-β promoter-driven luciferase activity 7.9- and 103-folds, respectively . IE86 protein significantly reduced both STING- and TRIF-induced IFN-β promoter activation, possibly by inhibiting NF-κB activity as previously reported   , 2006. TFigure 9A). At 48 h after transfection, IFN-β promoter activities and the level of STING protein were assessed by luciferase reporter assay and western blot analysis, respectively . Expression of full-length or truncated IE86 protein containing amino acids (aa) 1-290, 1-542, 86-542 or 136-579 significantly reduced STING-induced IFN-β promoter activation . On the other hand, expression of IE86 mutant protein containing aa 290–542 or 543–579 failed to exhibit significant inhibitory effect on STING-induced IFN-β promoter activation . Expression of full-length or truncated IE86 protein containing aa 1–290, 1–542, or 136–579 significantly reduced the level of STING protein in HEK293T cells . However, expression of IE86 mutant protein containing aa 86–542, 290–542, or 543–579 failed to reduce the level of STING protein in HEK293T cells . Taken together, aa 1-289 of the IE86 protein may be critical for down-regulating both STING-induced IFN-β promoter activation and the level of STING protein.To further elucidate the IE86 protein residues that are required for inhibition of STING-induced IFN-β promoter activation and down-regulation of STING protein levels, HEK293T cells were co-transfected with expression vectors for STING and myc-tagged IE86 WT or deletion mutants (The type I IFN pathway plays an important role in limiting HCMV replication. Pre-infection or post-infection treatment of type I IFN significantly inhibits virus production . FurtherDNA sensors such as DNA-dependent activator of IFN-regulatory factors (DAI), IFI16 or cGAS were reported to detect HCMV infection to induce type I IFN activation . Since CFigure 1A), whereas STING KD exhibited more profound effect on HCMV infection in HUVEC cells   . These dFigure 4A)  . Howevergure 4A) . The difFigures 7A).Given the importance of STING in the innate immune response against DNA viruses, it is not surprising that 7 viral genes  have been identified in HCMV cDNA library screening to significantly down-regulate the STING-induced IFN-β promoter activation. Although additional enhancement of STING-induced IFN-β promoter by UL76 and UL50 is noteworthy, the importance of IFN-β promoter enhancement by these viral proteins in the context of HCMV life cycle is unclear. HCMV UL76 protein was reported to induce IL-8 expression via NF-κB activation . Thus, SFigure 3) and (ii) strongly interacted with STING in the yeast two hybrid screening to identify STING-interacting HCMV ORFs . Given the fact that IE86 protein is predominantly localized in the nucleus, it was unexpected that IE86 protein interacts with STING in the yeast two-hybrid assay. In addition, we were unable to determine the interaction between IE86 and STING in HCMV-infected HFF cells with or without MG132 treatment (data not shown). Since MG132 affects the MIE promoter and/or may induce adverse effects on cells  reduced STING-induced IFN-β promoter activation most effectively among the screened HCMV ORF . Since UL82 is critical for efficient expression of IE genes . Therefore, interaction between UL82 protein and STING may be indirect and involve other cellular proteins such as iRhom2  and tripartite motif protein 30α (TRIM30α) catalyze lysine 48-linked polyubiquitination and proteasome-dependent degradation of STING . The IE86 aa 136 to 290 region is rich in serine and threonine residues which is highly phosphorylated and is critical for interaction with various host factors such as Retinoblastoma Protein (Rb) or TATA-binding protein (TBP) . Since IE86 protein regulates gene expression by interacting with numerous viral and cellular proteins  . HPV E7 in (TBP) . Althougin (TBP) , the funJ-EK and Y-JS designed experiments, analyzed data, and wrote the manuscript. J-EK and Y-EK performed experiments. MS and J-HA provided materials. Y-JS, MS, and J-HA revised 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.


In response to parathyroid hormone (PTH), a bone anabolic hormone, LepR+Runx2-GFPlow cells increase Runx2 expression and form multilayered structures near the bone surface. Subsequently, the multilayered cells express Osterix and Type I collagen α, resulting in generation of mature osteoblasts. Therefore, our results indicate that Runx2 is weakly expressed in the LepR+ population without osteoblastic commitment, and the LepR+Runx2-GFPlow stromal cells sit atop the BM stromal hierarchy.Bone marrow mesenchymal stem and progenitor cells (BM-MSPCs) maintain homeostasis of bone tissue by providing osteoblasts. Although several markers have been identified for labeling of MSPCs, these labeled cells still contain non-BM-MSPC populations. Studies have suggested that MSPCs are observed as leptin receptor (LepR)-positive cells, whereas osteoblasts can be classified as positive for Runx2, a master regulator for osteoblastogenesis. Here, we demonstrate, using Runx2-GFP reporter mice, that the LepR-labeled population contains Runx2-GFP The clonally expanded CFU-F colonies and mesenspheres have differentiation potential to osteoblasts, adipocytes and chondrocytes both in vitro and in vivo. BM-MSPCs can be marked by the expression of leptin receptor (LepR)-Cre, and are distributed nearby blood vessels throughout the whole BM cavity6. In vivo fate mapping approaches demonstrated that LepR+ cells differentiate to osteoblasts and adipocytes under normal conditions. The contribution of LepR+ cells to chondrocytes is observed during the healing process of bone tissue6. There is evidence that LepR-Cre-labeled cells largely overlap with other markers for the BM-MSPC populations including CD31−CD45−Ter119−Nestin-GFPlow cells7, CXCL12 abundant reticular (CAR) cells9, PDGFRβ+ cells6 and Prx-1-Cre labeled cells10. Although these markers make it possible to enrich the BM-MSPCs from whole BM cells, not all the labeled cells have the potential to form CFU-F colonies or clonal mesenspheres11. These results suggest that the fractions are impure and still contain non-BM-MSPC populations.Bone marrow (BM) cells belonging to mesenchymal lineages are derived from mesenchymal stem and progenitor cells (MSPCs). BM-MSPCs are traditionally characterized as cells possessing colony forming potential in adherent culture conditions  and have the ability to form clonal spheres in nonadherent culture conditions [designated as mesenspheres]14. Osteoblastogenesis is fully suppressed by the global knockout of Runx214. Exon 8 of Runx2 gene conditional deletion in mature osteoblasts, which express Cre recombinase under the control of a 2.3-kb fragment of the type I collagen α[(Col1(2.3)] promoter, exhibit low bone mass phenotype15. In contrast, conditional knockout mice lacking exon 4 of Runx2 gene in mature osteoblasts have no effect on osteoblastic activity16. These studies indicate that the necessity of Runx2 in osteoblastic activity is still controversial. On the other hand, in vivo lineage tracing studies have demonstrated that Runx2 is essential for osteoblast lineage commitment17. Interestingly, Runx2 overexpression approaches revealed that the late stage of osteoblastogenesis is negatively regulated by Runx2, whose levels were found to decrease with osteoblast maturation19. Overall, these findings suggest that Runx2 is required for osteoblast commitment from immature mesenchymal stromal cells. These results raise the intriguing possibility that Runx2 may be expressed in a portion of LepR+ stromal cells, which have osteogenic-committed sub-populations.Runt-related transcription factor 2 (Runx2) is a master regulator for osteoblast differentiation20. Furthermore, bone formation is inhibited by conditionally deleting Osx in mature osteoblasts21. These results suggest that Osx is necessary not only for osteoblast differentiation, but also for their functions. On the other hand, during endochondral bone ossification, BM-MSPCs are generated from part of the developing chondrogenic cell populations17. The expression levels of Osx are increased throughout the development of chondrogenic cell populations that subsequently differentiate into BM-MSPCs22. Although Osx protein expression in BM-MSPCs is completely lost in the adult stage, mRNA expression is maintained23. However, the Osx expression pattern during osteoblastogenesis from BM-MSPCs has yet to be elucidated.Osteoblastogenesis is completely diminished in knockout mice lacking Osterix (Osx), a transcription factor that acts downstream of Runx224. Several studies have demonstrated that intermittent PTH treatment induces remedial action against osteoporosis due to anabolic effects on bone tissue28. Researchers have found that osteoblast precursors are increased along the bone surfaces in response to PTH treatment30. These results suggest that the anabolic effects of PTH on bone tissue are exerted by the acceleration of osteoblastogenesis from immature BM mesenchymal precursors. However, it still remains unclear which BM stromal cells give rise to osteoblasts in response to PTH treatments, thereby mediating the therapeutic response in osteoporosis.Teriparatide, a biologically active amino acid 1–34 fragment of human PTH [hPTH (1–34)], is clinically used in treatment of osteoporosis patients+ cell population contains Runx2-GFPlow cells, and unexpectedly, that stem cell capacity is enriched in the Runx2-GFPlow sub-population. In addition, our studies have shown that the LepR+Runx2-GFPlow cells differentiate into mature osteoblasts via multilayered cell formation adjacent to bone surfaces in response to PTH-induced bone anabolic effects. These results provide evidence that LepR+Runx2-GFPlow cells sit atop the BM mesenchymal stromal cell hierarchy.Here we demonstrate, using Runx2-GFP reporter mice, that the LepR31. FITC-conjugated anti-GFP antibody was used to amplify the intensity of the GFP signal when imaging bone tissue sections. Consistent with a previous study31, Runx2-GFP+ expression in bone tissues was detected in osteoblasts, osteocytes and chondrocytes  BM stromal cells, which are considered to have characteristics of BM-MSPCs, also weakly expressed Runx2-GFP throughout the bone marrow cavity  of Runx2 locustes Fig. . Interestes Fig. . On the tes Fig. . It is iity Fig. . These r+ cells were observed in the BM stromal cell population without enhancing the GFP signal by FITC-conjugated anti-GFP antibody  , but not Runx2-GFPhigh cells (3.3 ± 1.8%), were positive for LepR  (designated as LepR+Runx2-GFPlow cells)  Fig. . Most ofepR Fig. . Previoulls Fig. . These r32. We next generated LepR-Cre/ROSA26-loxP-stop-loxP-tdTomato/Runx2-GFP (LepR/Tomato/Runx2-GFP) mice, and analyzed whether stem cell activity in LepR+ cells increases in inverse proportion to Runx2 expression levels. Previous reports demonstrated that the differentiation of LepR+ cells into mature osteoblasts is mainly observed in adult mice, and only rarely seen in young mice6. In order to avoid contamination of the LepR-Cre-labeled population with LepR+ cell-derived osteoblasts, we analyzed young mice (5–7 weeks old) in this experiment. Histological analysis demonstrated that bone-lining osteoblasts were strongly positive for Runx2-GFP, but negative for LepR/Tomato  cells do not contain the osteoblastic population in young mice. On the other hand, the LepR/Tomato+ cells in the BM cavity were positive for Runx2-GFP   at greater frequency than other fractions  cells23, which are generated from part of the developing chondrogenic cell populations17. The expression levels of Runx2 mRNA in the Runx2-GFPlow sub-population were significantly higher than those in the Runx2-GFP− sub-population from LepR-Cre/Tomato+ stromal cells   Fig. . We thenons Fig. . Consistion Fig. . Contrarigh Fig. . CFU-F cive Fig. . Furtherons Fig. . The Lepial Fig. . However223Fig. . Interesion Fig. . These rlls Fig. . The Runvel Fig. . ConsistIn vivo genetic lineage tracing analysis demonstrated that LepR+ cells differentiate into osteoblasts6. As we found that stem cell activity is enriched in LepR+Runx2-GFPlow BM stromal cell populations, we next examined whether LepR+Runx2-GFPlow cells differentiate into mature osteoblasts in vivo by lineage tracing. Because intermittent treatment of parathyroid hormone (PTH) (1–34) increased bone volume by inducing bone formation  were observed in the vicinity of bone tissue by PTH treatment /Tomato/Runx2-GFP mice, administering tamoxifen for labeling of Osx+ cells34. Histological analyses demonstrated that mature osteoblasts express both iOsx/Tomato and Runx2-GFP, but Runx2-GFPlow cells located away from bone surfaces were negative for iOsx/Tomato  were significantly increased by the PTH treatment , a transcription factor downstream of Runx2, in ML-cells using Osx-Cre+ cells were observed as mature osteoblasts with Col1(2.3)-GFP expression in control bone tissue  were significantly increased by the PTH treatment , a marker for mature osteoblasts, in osteoblastogenesis from ML-cells using iOsx/Tomato/Col1(2.3)-GFP mice. Tamoxifen-induced iOsx/Tomatoent Fig. . Only thsts Fig. .Figure 5in vivo osteoblastogenesis from LepR+ cells, we analyzed the expression pattern of the early osteoblastic transcription factor, Runx2, in LepR+ cells using Runx2-GFP transgenic mice31. Here we report that BM-MSPCs in adult BM are confined to the weak Runx2-GFP-expressing LepR+ stromal cell population, which differentiates into Col1+ mature osteoblasts in response to PTH anabolic effects. In this process, the LepR+Runx2-GFPlow cells form multilayered structures along the bone surface, subsequently increasing expression levels of Runx2 and Osx.To clarify low populations are consistent with recently published data in which the most primitive stromal population in the calvaria, gated as Prx1+Scal+ cells, expresses Runx2 at low levels35. On the other hand, a single-cell assay demonstrated that CXCL12 abundant reticular (CAR) cells, which largely overlap with the LepR+ cell population, express not only Runx2 and Osx, but also peroxisome proliferator-activated receptor γ (PPARγ), an essential transcription factor for adipogenesis36, at the mRNA level23. Interestingly, osteoblastogenesis is enhanced by decreased transcriptional activity and haploinsufficiency of PPARγ in BM stromal progenitors38. In contrast, adipogenesis is accelerated due to the stromal deletion of Wnt/β-catenin-signaling, an essential signaling pathway for osteoblastogenesis39. These reports suggest that the undifferentiated state of LepR+Runx2-GFPlow cells is sustained due to reciprocal inhibition between osteogenic and adipogenic factors. Several studies have reported that the lineage differentiation of BM-MSPCs is skewed toward osteoblasts by intercellular expression of vascular endothelial growth factor A (VEGF-A)40. In contrast, the adipocyte lineage commitment from BM-MSPCs is increased by up-regulation of MicroRNA-18841, deletion of transcription factor Foxc19 and peripheral Leptin/Leptin receptor signaling10. Further studies will clarify the mechanistic details of the cell fate decision of BM-MSPCs.Our results demonstrating that the stem cell capacity is enriched in Runx2-GFP− stromal sub-population of LepR+ cells. These results indicate that the LepR+Runx2-GFP− stromal sub-population contains some kind of committed cells other than stem cells. Previous studies provide in vivo evidence demonstrating that LepR+ cells differentiate into not only osteoblasts but also adipocytes with aging and during tissue regeneration processes after injury6. In the process of adipogenesis, the expression levels of Runx2 decrease, as opposed to the increase of PPARγ40. Furthermore, our immunofluorescence data demonstrated that adipocytes are negative for Runx2 , Runx2, osteocalcin and osteonectin30. These results indicate that the PTH-induced osteoblastic differentiation is associated with cell cycle progression. Previous studies reported that the LepR+ cells are quiescent in adult BM6. However, the LepR+ cells markedly proliferate in response to self-depletion, and lineage differentiation into both osteoblasts and adipocytes is accelerated concomitantly in this situation6. In contrast, depletion of the transcription factors Snail and Slug in the skeletal stem cells decreases not only proliferative activity but also lineage commitment potential, coincidently44. These results suggest that cell cycle quiescence may be critical for maintaining the undifferentiated state of BM-MSPCs. Further analysis of the mechanistic relationship between cell cycle regulation and lineage commitment of LepR+Runx2-GFPlow cells will provide a potential therapeutic target for osteoporotic patients.In this study, we demonstrated that the PTH treatment accelerated osteoblastogenesis from LepRtm2(cre)Rck/J (LepR-Cre), B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mice were purchased from Jackson Laboratory. Osx-CreERT2 mice34 were provided from H.M. Kronenberg. Col1(2.3)-Gfp mice45 and Runx2-Gfp mice31 were generated in one of the author’s laboratories, and backcrossed with C57BL/6 for 5 generations. 5–7 week-old mice were used for all experiments. All mice were maintained in pathogen-free conditions in animal facilities certified by the Animal Care and Use Committees of Matsumoto Dental University, and animal protocols were approved by that committee. All animal studies were performed in accordance with the Guidelines of the Matsumoto Dental University Animal Care Committee.C57BL/6, B6.129-LeprThe primary antibodies used were Alexa Fluor 647-anti-VE-Cadherin and Alexa Fluor 647-anti-CD31/PECAM-1 (MEC13.3) ; APC or PE-anti-CD45 (30-F11), APC or PE-anti-Ter119 (Ter119) ; anti-LepR, anti-SOST/Sclerostin and anti-fatty acid binding protein 4 (FABP4) ; anti-Osteocalcin (R21C-01A) and anti-DMP-1 ; anti-Perilipin ; anti-chick typeII collagen (A2-10) (Chondrex); anti-Runx2 (D1L7F) . The secondary antibodies used were Alexa Fluor 647 donkey anti-goat IgG and Alexa Fluor 594 donkey anti-rat IgG ; Cy3 donkey anti-mouse IgG (Merck Millipore); FITC donkey anti-rabbit IgG (Bethyl Laboratories). Alexa Fluor 488-anti-GFP (Molecular Probes) was used for enhancement of the Runx2-GFP signal. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich) or TO-PRO-3 Iodide (642/661) (Molecular Probes).ERT2 mice, CRF-1 chow diet  containing tamoxifen (Sigma-Aldrich) at 400 mg/kg was given from 5 days before the first PTH injection until the end of the experiment.Human PTH (1-34) was kindly provided from Asahi Kasei Pharma Co. Ltd. Four-six-week-old mice were intraperitoneally injected with PTH (80 μg/kg/12 hours) for 10 days. Forty-eight hours after the final PTH injection, mice were sacrificed and used for analyses. For induction of Cre-mediated recombination in Osx-Cre46. Z-stack confocal projection images were obtained from 2-μm interval images from 10–20-μm thick sections. Fluorescence and phase-contrast images were acquired using a laser-scanning confocal microscope  equipped with Plan-Apochromat (10×/0.45 and 20×/0.8), ZEN and Axiovision software (Carl Zeiss). Bright-field images were acquired using a Light microscope Zeiss Axiovert 200 (Carl Zeiss) equipped with Plan-NEOFLUAR (2.5×/0.075), LD A-plan (40×/0.50 Ph2) and Axiovision software (Carl Zeiss) and Stemi 2000-C (Carl Zeiss).Mice were perfused with 4% paraformaldehyde (PFA) for fixation, and bone tissue were further fixed with 4% PFA for 24 hours at 4 °C, and incubated in 10%, 20% and 30% sucrose each for 2 hours at 4 °C for cryoprotection, then embedded in 5% carboxymethyl cellulose (SECTION-LAB). Sections, 10–20-μm thick, were prepared using Kawamoto’s film method47. BM was digested with 0.1% collagenase IV (Gibco), 0.2% Dispase (Gibco) and 20 U/ml DNase  in HBSS (Gibco) for 30 min at 37 °C.BM was gently flushed in L-15 FACS buffer3 cells per well in a 12-well adherent tissue culture plate using a MesenCult proliferation Kit with MesenPure (StemCell Technologies) containing 100 U/ml and 100 μg/ml penicillin-streptomycin. Half of the media was replaced after 7 days and at day 10, cells were stained with Giemsa staining solution  and adherent colonies were counted.Mouse sorted cells were seeded at 2–3 × 103 were transferred to non-adherent 24 well plates (Corning) with spheroid-forming media47 . After 7 days, the spheroid efficiency was determined.Mouse sorted cells at 1 × 10Cell sorting experiments were performed using an Aria III Cell Sorter (BD Biosciences). Flow cytometric analyses were carried out using a Cytomics FC 500 flow cytometer equipped with CXP software . Dead cells and debris were excluded by FSC, SSC, DAPI (Dojindo) and Fixable Viability Dye eFluor 780 (eBioscience) staining profiles. Data were analysed with FlowJo (Tree Star) software.+Runx2-GFPlow stromal cells were expanded using a MesenCult proliferation Kit with MesenPure (StemCell Technologies) containing 100 U/ml and 100 μg/ml penicillin-streptomycin. Osteogenic, adipogenic and chondrogenic differentiation were induced using a Mouse Mesenchymal Stem Cell Function Identification Kit (R&D Systems). Cells were maintained with 5% CO2 in a water-jacketed incubator at 37 °C for 2–5 weeks. Mineralized osteogenic cells were identified by Alizarin Red S (Sigma-Aldrich) staining. Adipocytes were identified by characteristic production of lipid droplets and staining with an anti-FABP antibody (R&D Systems). Chondrocytic cells were identified using an Alcian Blue 8GX solution (Sigma-Aldrich).Sorted LepR-Cre/TomatoTM (Applied Biosystems). Gene expression data was normalized to Gapdh. The sequences of primers for each gene were as follows: Gapdh, 5′-TGTGTCCGTCGTGGATCTGA-3′ (forward) and 5′-TTGCTGTTGAAGTCGCAGGAG-3′ (reverse); Runx2 (Type II isoform), 5′-CCAGCCACCGAGACCAACC-3′ (forward) and 5′-GTTTGACGCCATAGTCCCTCC-3′ (reverse); LepR, 5′-TCAGAATTTTGGGTGGAAAA-3′ (forward) and 5′-GTCCAGGTGAGGAGCAAGAG-3′ (reverse); PDGFRa, 5′-AGCAAACATCTTGACTTGGGAACA-3′ (forward) and 5′-ACTTGCATCATTCCCGGACAC-3′ (reverse); CXCL12, 5′-CCAGAGCCAACGTCAAGCAT-3′ (forward) 5′-CAGCCGTGCAACAATCTGAA-3′ (reverse).Sorted cells were collected in TRIzol reagent (Ambion) and mRNA was purified using a PureLink RNA Micro kit (Invitrogen). Reverse transcription and quantitative real-time PCR were performed using a One Step SYBR Prime Script PLUS RT-PCR kit (TAKARA) and an Applied Biosystems StepOnePlusFemora were fixed in 70% ethanol. Three-dimensional (3D) reconstructions of distal femora were obtained by micro-computed tomography (μCT) . Morphological indices were calculated in trabecular bones located from 0.5 to 1.5 mm from the growth plates using image analysis software .t-tests. Experiments were performed three times and similar results were obtained. Statistical analyses were performed with Graph Pad Prism 6. P < 0.05 was considered significant.The results were expressed as mean ± SD. Data were evaluated by unpaired Student′s Supplementary Information


DCTPP1-knockdown GC cell line BGC-823 exhibited more sensitivity to 5-fluorouracil (5-FU), demonstrated by the retardation of cell proliferation, the increase in cell apoptosis, cell cycle arrest at S phase and more DNA damages. Multidrug resistance 1 (MDR1) expression was unexpectedly down-regulated in DCTPP1-knockdown BGC-823 cells together with more intracellular 5-FU accumulation. This was in large achieved by the elevated methylation in promoter region of MDR1 gene. The intracellular 5-methyl-dCTP level increased in DCTPP1-knockdown BGC-823 cells as well. More significantly, the strong correlation of DCTPP1 and MDR1 expression was detectable in clinical GC samples. Our results thus imply a novel mechanism of chemoresistance mediated by the overexpression of DCTPP1 in GC. It is achieved partially through decreasing the concentration of intracellular 5-methyl-dCTP, which in turn results in promoter hypomethylation and hyper-expression of drug resistant gene MDR1. Our study suggests DCTPP1 as a potential indicative biomarker for the predication of chemoresistance in GC.Gastric cancer (GC) is among the most malignant cancers with high incidence and poor prognoses worldwide as well as in China. dCTP pyrophosphatase 1 (DCTPP1) is overexpressed in GC with a poor prognosis. Given chemotherapeutic drugs share similar structures with pyrimidine nucleotides, the role of DCTPP1 in affecting the drug sensitivity in GC remains unclear and is worthy of investigation. In the present study, we reported that  Gastric cancer (GC) is of great threat to human worldwide due to its high incidence and mortality among cancers , 2. In CEscherichia coli [Mycobacterium tuberculosis can safeguard genetic stability via degrading 5-OH-dCTP [Nucleoside triphosphate pyrophosphatases (NTP- PPases) belong to nuclease family that are capable of hydrolyzing α-β phosphodiester bond of (d)NTPs into corresponding monophosphate and pyrophosphoric acid (PPi) . To datehia coli , 9. MazG-OH-dCTP . Certain-OH-dCTP , ITPase -OH-dCTP  and MTH1-OH-dCTP , 14. Mor-OH-dCTP –18, pote-OH-dCTP , 19, 20.dCTP pyrophosphatase 1 (DCTPP1) is an NTP-PPase newly identified in human whose structure contains a bacterial MazG domain . It hydrIt is previously reported that the putative DCTPP1 inhibitors enhance the cytotoxicity against leukemia cells, including 5-azacytidine, decitabine, and gemcitabine . ConsideDCTPP1 stable knockdown BGC-823 cells (BGC-823-shRNA1 and BGC-823-shRNA2) by transfecting vectors containing short hairpin RNA (shRNA) specific to DCTPP1  of 5-FU when compared to BGC-823-NC cells   Figure . More cl) Figure . These rDCTPP1 alone had little effect on cell cycle arrest in BGC-823 cells, which was consistent with the results from proliferation assay   Figure . The incs Figure . These fDCTPP1-knockdown and control BGC-823 cells without 5-FU treatment, more AP sites were detected in two DCTPP1-knockdown BGC-823 cells than control cells after 5-FU treatment even at low concentrations  and anti-apoptotic genes   and functions as an ATP-dependent drug efflux pump that reduces intracellular concentrations of chemotherapeutic agents including 5-FU [MDR1 expression in DCTPP1-knockdown and control BGC-823 cells. It was shown that both the mRNA and protein levels of MDR1 significantly decreased in DCTPP1-knockdown BGC-823 cells when compared to control cells  is a classical protein responsible for multi-drug resistance . It is eing 5-FU . Therefos Figure  and 6B. s Figure . Moreoveh Figure . Our resMDR1 is reported to be due to CpG hypermethylation in promoter region in certain cancers [MDR1 gene between BGC-823-NC and BGC-823-shRNA cells. 20 CpG islands in MDR1 gene promoter region were subjected to methylation analysis by using a bisulfite sequencing PCR approach. 10 clones for each CpG island were randomly selected for sequencing    Figure , whereas= 0.068) . InteresDCTPP1 significantly increased the sensitivity to 5-FU in GC cell line BGC-823 cells. Moreover, low expression of DCTPP1 led to the increase in intracellular 5-methyl-dCTP, which was strongly associated with the promoter hyper-methylation, leading to the subsequent low-expression of MDR1 and the increased intracellular accumulation of 5-FU in DCTPP1-knockdown BGC-823 cells. These results provide new insights into the roles of DCTPP1 as a chemosensitizer in clinical application.DCTPP1 is the only dCTP pyrophosphatase identified in human targeting dCTP and its structure derivates. Recently, its biological function and clinical significance have been illustrated , 22. In DCTPP1-knockdown and control BGC-823 cells. These results impel us to focus on another conventional mechanism of drug resistance mediated by drug transportation.5-FU is one of the first-line chemotherapeutic drugs against GC. However, the overall efficacy of 5-FU treatment is somehow limited in clinic due to drug resistance . The molMRP1 and MXR/BCRP had no significant changes between DCTPP1-knockdown and control BGC-823 cells  and mitoxantrone resistance protein (MXR/BCRP), are the major cause for chemotherapy failure . Our res23 cells . But thes Figure  and 6B. s Figure . This leDCTPP1 knockdown affects MDR1 expression remains ambiguous. In some carcinomas, the CpG hypermethylation of MDR1 promoter is thought to be one of the mechanisms causing down-regulation of MDR1 [MDR1 promoter region significantly increased after DCTPP1 knockdown. It is well documented that DNA methylation is an enzyme-mediated post-replication process where DNA methyltransferase (DNMT) plays a critical role [DCTPP1-knockdown and control BGC-823 cells. But the expressions of DNMT1, DNMT3A and DNMT3B had no significant difference before and after DCTPP1 knockdown in BGC-823 cells  and routinely maintained in DMEM medium  supplemented with 10% fetal bovine serum (FBS) (Gibco) in a 5% CORabbit anti-human DCTPP1 polyclonal antibody (pAb) (Cat#AP2821a) was obtained from Abgent . Mouse anti-human MDR1 (Cat# sc-55510) and mouse anti-DNMT1 monoclonal antibody (mAb) (Cat# sc-271729) were purchased from Santa Cruz Biotechnology . Mouse anti-α-tublin Ab (Cat# T6074) was purchased from Sigma . Rabbit anti-Phospho-BRCA1 Ab (Cat# 9009), rabbit anti-Phospho-H2A.X Ab (Cat# 2197), rabbit anti-caspase-3 (8G10) mAb (Cat# 9665), rabbit anti-cleaved caspase-3 (Asp 175) mAb (Cat# 9664), rabbit anti-Bax pAb (Cat#2772), rabbit anti-Bcl-2 (50E3) mAb (Cat# 2870), HRP-linked anti-rabbit IgG Ab (Cat# 7074) and HRP-linked anti-mouse IgG Ab (Cat# 7076) were purchased from Cell Signaling Technology . dCTP, dTTP and 5-FU were purchased from Sigma and 5-methyl-dCTP was purchased from New England BioLabs .DCTPP1-knockdown BGC-823 cells were constructed by transfecting RNAi-Ready pSIREN-RetroQ retroviral vector  containing shRNA oligonucleotides targeting DCTPP1 as described in our previous report [DCTPP1 and NC sequences were listed in Table Stable s report . BrieflyDCTPP1-knockdown BGC-823 cells, DCTPP1 gene was amplified by PCR with the template from Hela cDNA (primer sequences were listed in Table 5/2 mL) were seeded in 6-well plates and transfected with recombinant plasmid pIRESpuro-DCTPP1 or empty vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. BGC-823 cells were trypsinized, counted and reseeded in 96-well plates for another 24 h after transfection. Cells were cultured with 5-FU (Sigma) at different concentrations  for 72 h and subjected to cell viability assay.To rescue DCTPP1 in 3/100 μL) were seeded in 96-well plates . After 24 h, cells were cultured with 5-FU (Sigma) at different concentrations  for 72 h. Cell viability was analyzed afterwards by using CCK-8 kit  according to the manufacturer's instructions. The absorbance was measured at 450 nm on a microplate reader . IC50 (72h) value was calculated by GraphPad Prism 5 software .Cells  for 72 hours. Cell viability was analyzed by using CCK-8 assay.6 BGC-823-NC or BGC-823-shRNA1 cells. When the volumes of xenograft tumors reached 180-200 mm3, mice were randomly subdivided into two groups with 5 mice in each group. Experimental groups were treated with 5-FU intraperitoneally (25 mg/kg) at every other day for two weeks while control groups were treated with PBS. The volumes of xenograft tumors were measured by caliper and calculated by the following formula V = 1/2 × (length × width2). After 4 weeks, nude mice were sacrificed and xenograft tumors were harvested and weighed. The protocols of animal experiments were approved by the Animal Ethics Committee of SJTUSM, and were performed under the Guide for the Care and Use of Laboratory Animals.Six-week-old BALB/c nude mice  were subcutaneously injected with 3 × 105/2 mL) were seeded in 6-well plates and treated with or without 100 μM 5-FU. After incubation for 48 h, total cells were harvested and washed twice with cold PBS. Cell apoptosis was analyzed by using FITC-Annexin V Apoptosis Detection Kit  according to the manufacturer's instructions. Cells were acquired by FACS Calibur flow cytometer  and data analysis was performed by using FlowJo 7.6 software .Cells (1 × 105/2 mL) were seeded in 6-well plates and treated with or without 1 μM 5-FU. After incubation for 48 h, adherent cells were trypsinized, collected and washed twice with cold PBS. Cell pellets were resuspended in 5 mL ice-cold PBS containing 75% ethanol and fixed at -20°C overnight. Cells were stained with PI/RNase staining buffer  according to DNA staining protocol for flow cytometry. Cells was acquired by FACS Calibur flow cytometer (BD Pharmingen) and cell cycle was analyzed by ModFit software .Cells (1 × 105/2 mL) were seeded in 6-well plates and treated with or without 5-FU. After incubation for 48 h, adherent cells were collected. Proteins from some of the cells were extracted by RIPA lysis buffer. DNA damage markers, phospho-BRCA1 and phospho-H2A.X, were measured by Western blot. In addition, chromosomal DNA was extracted from part of the cultured cells. AP sites were measured using DNA Damage Quantification kit (Dojindo Laboratories) according to the manufacturer's instructions.Cells  . The whole cell extracts were collected and protein concentration was determined by BCA protein assay kit . Cell lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes . The membranes were blocked with PBS (pH7.4) containing 5% non-fat milk for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C. After incubation with HRP-conjugated secondary antibodies for 1 h, immunoreactive proteins were visualized with ECL detection system (Millipore Co.). Intensities of the bands were quantified by using an image analysis system (BioRad).® Premix Ex Taq™ kits  according to the manufacturer's instructions.Total RNA was extracted with Trizol reagent  according to the manufacturer's instructions. First-strand cDNA was synthesized by using a PrimeScript™ RT reagent Kit with gDNA Eraser . The primer sequences used in real-time PCR were listed in Table MDR1 gene including 20 CpG dinucleotides that have been reported to be highly linked to the regulation of MDR1 expression [2, 0.2 μM primer, 12.5 units TaqHS (Takara Biotechnology), and 600 ng bisulfite-modified DNA. The BSP conditions were 94°C for 1 min, followed by 37 cycles at 94°C for 15 s, 54°C for 20 s, and 72°C for 25 s, with a final extension at 72°C for 10 min. PCR products were purified and cloned into the pEASY-T1 vector for sequencing. For each PCR sample, 10 individual clones were randomly selected and sequenced in the experiments. In the BSP assay, the treatment of bisulfite led to the conversion of cytosine residues into uracil whereas 5-methyl-cytosine residues unaffected. After PCR amplification, original unmethylated cytosines were converted to thymine while methylated cytosines remained. Therefore, methylation level was determined according to the average methylation rate of 20 CpG dinucleotides for each DNA sample.Total DNA was extracted from cultured cells using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit (Takara Biotechnology). Bisulfite sequencing PCR (BSP) was performed in BioSun Biotech Co. Ltd. . Briefly, MDR1-BSP-F and MDR1-BSP-R primers were designed for the amplification of the promoter region of pression –38. The 6 cells were extracted with 500 μL pre-cold 60% methanol and immediately frozen in liquid nitrogen. After storage at −80°C for at least 24 h, sample solutions were centrifuged at 14,000 g for 10 min at 4°C. The supernatants were collected and stored at −80°C until for LC-MS/MS analysis.Intracellular nucleotides were measured by LC-MS/MS as described in our previous study  with min6 BGC-823 cells were seeded in 10 cm dish overnight and treated with 10 mL DMEM medium containing 100 μM 5-FU. After incubation for 1 h, the medium was replaced with 10 mL DMEM medium. After incubation for another 15 min, the intracellular 5-FU was extracted by pre-cold 60% methanol as described above.Intracellular 5-FU was measured by LC-MS/MS as described in previous report  with minLC-MS/MS assay was performed using an Agilent 1200 HPLC system coupled to an Agilent 6410 triple quadruple mass spectrometer . The mode of multiple reaction monitoring (MRM) was used to identify and quantify dCTP , 5-methyl-dCTP , dTTP , and 5-FU . The data of peak areas were acquired and the relative quality of compound tested in BGC-823 cells was analyzed accordingly to their areas of peaks.Tissue microarray containing 30 formalin-fixed, paraffin-embedded GC tissues and 30 paired adjacent tissues (2.0 mm in diameter and 4 μm in thickness) was purchased from the Shanghai Outdo Biotech Company . The expressions of DCTPP1 and MDR1 in GC tissue microarray were tested by immunohistochemistry described in our previous report using anti-DCTPP1 and anti-MDR1 antibodies . Slides t-test with SPSS version 20.0 . Differences between groups were determined as statistically significant at P < 0.05. The correlation between DCTPP1 and MDR1 expression was assessed using Pearson correlation coefficient. P < 0.05 was considered significant.All the data were represented as the means ± standard deviation (SD). Statistical analysis was carried out by using two-tailed Student's


Caenorhabditis elegans during which the epidermal apical extracellular matrix, termed the cuticle, is detached and replaced at each larval stage. The conserved NIMA-related kinases NEKL-2/NEK8/NEK9 and NEKL-3/NEK6/NEK7, together with their ankyrin repeat partners, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, are essential for normal molting. In nekl and mlt mutants, the old larval cuticle fails to be completely shed, leading to entrapment and growth arrest. To better understand the molecular and cellular functions of NEKLs during molting, we isolated genetic suppressors of nekl molting-defective mutants. Using two independent approaches, we identified CDC-42, a conserved Rho-family GTPase, and its effector protein kinase, SID-3/ACK1. Notably, CDC42 and ACK1 regulate actin dynamics in mammals, and actin reorganization within the worm epidermis has been proposed to be important for the molting process. Inhibition of NEKL–MLT activities led to strong defects in the distribution of actin and failure to form molting-specific apical actin bundles. Importantly, this phenotype was reverted following cdc-42 or sid-3 inhibition. In addition, repression of CDC-42 or SID-3 also suppressed nekl-associated defects in trafficking, a process that requires actin assembly and disassembly. Expression analyses indicated that components of the NEKL–MLT network colocalize with both actin and CDC-42 in specific regions of the epidermis. Moreover, NEKL–MLT components were required for the normal subcellular localization of CDC-42 in the epidermis as well as wild-type levels of CDC-42 activation. Taken together, our findings indicate that the NEKL–MLT network regulates actin through CDC-42 and its effector SID-3. Interestingly, we also observed that downregulation of CDC-42 in a wild-type background leads to molting defects, suggesting that there is a fine balance between NEKL–MLT and CDC-42–SID-3 activities in the epidermis.Molting is an essential process in the nematode  Caenorhabditis elegans. In this study, we found that NEK kinases regulate the organization of actin, a major component of the cytoskeleton and a player in many cellular processes. In the absence of normal NEK kinase activity, actin fails to form proper filamentous structures during worm development, which leads to growth arrest. Interestingly, these defects in nekl mutants can be partially reversed by simultaneously reducing the activities of CDC42 or ACK1, two conserved proteins that are key regulators of actin dynamics. Other findings suggest that NEK kinases control actin organization through functional interactions with CDC42 and ACK1. These studies provide a new link between NEK kinases and the actin cytoskeleton and, together with other reports, suggest that these functions may be conserved in humans.Protein kinases are key molecular regulators that act by modifying the structures and activities of proteins within the cell. Members of the NEK family of protein kinases regulate cell division and the formation of specialized organelles called cilia. Accordingly, mutations in the human NEK genes have been implicated in a number of diseases including cancer and maladies that result from defective cilia. To better understand the biological functions of NEK kinases, we have undertaken studies in the model system nematode,  Aspergillus nidulans, where it promotes cell cycle progression , 2625, 2nekl-2(fd91) and 79% (n = 53) of nekl-3(sv3) molting-defective larvae (nekl-2(fd91) and nekl-3(sv3) mutants, consistent with wild type, seam cells in these mutants were abnormally small and round relative to wild-type controls (mlt-3(RNAi) animals, including smearing and aggregation of the marker within the apical epidermis  of e larvae . In partcontrols , suggestpidermis . These fnekl and mlt mutants were due to indirect effects caused by improper shedding of the cuticle, we examined GFP::CDC-42 expression in several unrelated molting-defective backgrounds depleted for FBN-1, a fibrillin-like protein (fbn-1(tm290) mutants, as well as 71% (n = 14) of mlt-11(RNAi), and 100% (n = 10) of qua-1(RNAi) worms, indicating a specific role of the NEKL–MLT protein network in the control of CDC-42 localization.To address the possibility that the observed changes in GFP::CDC-42 localization in  protein ; MLT-11, protein ; or QUA- protein . We obsenekl function. As a dynamic switch, CDC-42 cycles between GTP-bound active and GDP-bound inactive forms. To assay CDC-42 activity, we made use of two independent reporters of CDC-42 activation. We first examined a GST::GFP::WSP-1(GBD) reporter that associates with GTP-bound active CDC-42 and is expressed under the control of the cdc-42 promoter , GR1395 [mgIs49(mlt-10::gfp-pest)], N2/Bristol (wild type), LH373 , NL2099 [rrf-3(pk1426)], RT1378 [pwIs528(gfp::chc-1)], SP2734 , SP2736 , WS5018 , WY1061 , WY1145 ; pTG96)]; WY1165 [nekl-2])], WY1217 , WY1242 , WY1331 , WY1332 , WY1333 , WY1337 , WY1338 , WY1345 , WY1352 , WY1353 , WY1360 ; pTG96)], WY1361 , WY1374 [sajIs31 ], WY1376 , WY1377 , WY1378 , WY1380 , WY1381 ; pTG96); sajIs31 ], WY1382 , WY1385 , WY1422 ; pTG96); xnIs506], and WY1424 .rotocols  and werecdc-42, we screened all available clones on LGII (~3000 clones) for clones that allowed nekl-3(sv3) mutants to bypass L2/L3 arrest. Control RNAi experiments were carried out using the bacterial strain HT115 carrying either a vector plasmid (pPD129.36) or gfp(RNAi). Marker localization studies involving mlt-3(RNAi) were carried out using strains that were previously grown for several generations on lin-35(RNAi) plates, which increases RNAi sensitivity [rrf-3(pk1426) mutants. Bacteria expressing mlt-2, mlt-3, cdc-42, and gfp(RNAi) constructs were grown to the same density and combined at specific ratios. A bacterial culture expressing mlt-2(RNAi) was combined at a 2:1 ratio with bacteria expressing cdc-42(RNAi) or gfp(RNAi), whereas the mlt-3(RNAi) bacterial strain was mixed with the above RNAi clones at a 1:2 ratio.RNAi was performed using bacterial strains from Geneservice using standard protocols . In the sitivity . Combinedpy-7 promoter region from plasmid pCFJ1662 (gift of Barth Grant) using the primers 5’- tcgacaaagctttctcattccacgatttctc-3’ and 5’- tcgacactgcagttatctggaacaaaatgtaagaa-3’. Following digestion with HindIII and PstI, PCR product was inserted into pD95.75 to create plasmids pDF400. Genomic cdc-42 coding sequences was amplified from fosmids WRM069cE02 and WRM0610aC01 using primers 5’-tgattcggatccatgcagacgatcaagtgcgtc-3’ and 5’-tgattcggtacctcgttccaattcacccactca-3’, digested with BamHI and KpnI, and ligated into PDF400 to produce pDF406. G12V and Q61L cdc-42 mutants were generated using the Q-5 Site-Directed Mutagenesis Kit (New England Biolabs) and the following primers: For G12V primers 5’-gttggagatgtagctgtcggtaaaac-3’ and 5’-gacgacgcacttgatcgt-3’; for Q61L primers 5’-actgctggactggaagattac-3’ and 5’- atcaaacaatcctaatgtgtatg-3’. G12V (pDF409) and Q61L (pDF412) Plasmids were sequence confirmed and injected with sur-5::GFP (pTG96) into 20–25 N2 worms at the following concentrations: 50 ng/μl sur-5::GFP + 100 ng/μl cdc-42, 100 ng/μl sur-5::GFP + 50 ng/μl cdc-42, 100 ng/μl sur-5::GFP + 20 ng/μl cdc-42, 100 ng/μl sur-5::GFP + 15 ng/μl cdc-42. Each injected worm was transferred to a new plate every 24 hours and its progeny were scored and imaged.Plasmids were generated by amplifying the sid-3(fd139) was identified as described in Joseph et al. [sid-3 in the nekl-2 (fd81); nekl-3(gk894345) background. Specifically, genome engineering was performed using CRISPR/Cas9 ribonucleoproteins in combination with the dpy-10 co-CRISPR method [sid-3. Absence of repair templates allowed for non-homologous repair mechanisms to generate random frameshifts and premature stop codons in the desired regions. Six loss-of-function alleles were isolated, including fd213, a 16-bp insertion and one point mutation in exon 13, causing a frameshift after L987 and premature stop at amino acid position 1034; fd214, a 7-bp deletion in exon 13, causing a frameshift after L987 and premature stop at amino acid position 1006; fd218, a 5-bp deletion in exon 1, causing a frameshift after K19 and premature stop at amino acid position 25; fd219, a 1-bp deletion in exon 1, causing a frameshift after A20 and premature stop at amino acid position 101; and fd221, a 31-bp insertion and 1-bp deletion in exon 1, causing a frameshift after A20 and premature stop at amino acid position 24  for 2 h at room temperature. Worms were washed three times in PBS and stored at 4°C. Phalloidin-TRITC  was diluted in permeabilization solution  at a ratio of 1:500. Equal amounts of liquid containing fixed animals and the phalloidin-containing solution were mixed and incubated in a dark chamber for at least 1 h. To avoid possible post mortem artifacts in our analyses, we scored only the animals that did not show any alterations in the morphology of their internal organs.Actin staining was performed following a modified procedure from Costa et al. . Animalsmlt-10p::gfp-pest reporter [Fluorescent images were acquired using an Olympus IX81 inverted microscope with a spinning-disc confocal head (CSUX1 Yokogawa). Confocal illumination was provided by an ILE-4 laser launch . MetaMorph7.7 software was used for image acquisition. DIC images were acquired using a Nikon Eclipse epifluorescence microscope and Open Lab software. This setup was also used for measuring body length and width. Body width was measured in the central region of animals; 50 animals were scored for each category. Animals were immobilized using a 0.1 M solution of levamisole. Distinguishing molting larvae from intermolt larvae was achieved using 0.5-μm red beads mixed with food (Sigma L3280), following the protocol of Nika et al. . For Figreporter , 102.z-stacks. Distances between neighboring planes in each z-stack were 0.2 μm. Composite sagittal plane projections were created from the same z-stacks in FIJI, using the Temporal-Color Code function.Fluorescence intensity of GFP::CDC-42 in S1 Fignekl-3(gk506) (A) and nekl-2(gk839) (B) animals grown on control RNAi (blue dots) and cdc-42(RNAi) (orange dots). Body width and body length are indicated in micrometers on the x and y axis, respectively. Each dot represents one animal (n = 50 for each genotype and RNAi treatment). A Student’s t-test was used to analyze differences in width. Graphic representation of body measurements of (TIF)Click here for additional data file.S2 Figrrf-3(pk1426) RNAi-hypersensitive animals treated with mlt-2(RNAi) (A) or mlt-3(RNAi) (B) combined with either control RNAi (blue dots) or cdc-42(RNAi) (orange dots). Each dot in graphs A and B represents one animal (n = 50 for each RNAi treatment). In both graphs, orange dots are typically shifted to the right side, indicating that cdc-42(RNAi) results in a body width increase. A Student’s t-test was used to analyze differences in width. Graphic representations of body measurements of (TIF)Click here for additional data file.S3 Figx axis represents fluorescence intensity (gray level) in arbitrary units; y axis represents distance from the starting point of line scan in micrometers. Each line scan starts at the upper left corner; direction of each line scan is indicated by the arrowhead. Bar size in C = 5 μm in A–C.(A-C) Line scans of selected green (NeonGreen) and red (mKate2) puncta in representative images from (TIF)Click here for additional data file.S4 Figmlt-3(RNAi) animals, resulting in the formation of large aggregates (white arrows).  Molting-defective mlt-11(RNAi) (C) and qua-1(RNAi) (D) animals display a relatively normal pattern of GFP::CDC-42 localization. Bar size in D = 10 μm in A–D.(A) GFP::CDC-42 is expressed in dispersed puncta throughout the epidermis in animals treated with a control RNAi. (B) GFP::CDC-42 is mislocalized in (TIF)Click here for additional data file.S5 Fignekl-2(fd91) (B) and nekl-3(sv3) (C) mutants, unlike in fbn-1(tm290) molting-defective controls (E). WSP-1(CRIB)::mCherry puncta are more numerous in nekl mutants (lower triangles in B and C) and sometimes form large aggregates (upper triangles in B and C). Bar size in D = 10 μm in A–D. (E) Quantitative comparison of average area of WSP-1(CRIB)::mCherry puncta measured in ten randomly selected animals for each indicated stage and genetic background. Error bars represent standard deviations. p values were derived using a Student's t-test.(A–H) Wild-type expression of a reporter for active CDC-42 (WSP-1(CRIB)::mCherry) (A) is changed in (TIF)Click here for additional data file.S6 Fig(A–K) Examples of molting defects in larvae expressing either the G12V or Q61L hyperactive variants of CDC-42 in the epidermis. DIC overlay  and accompanying GFP  images (panel K does not have an accompanying GFP image). DIC images are overlaid with GFP (colored magenta). Inset panels are indicated by colored boxes. Panels A–D were taken from the same animal. Abnormal cuticle is indicated by arrowheads. Bar sizes = 20 μm.(TIF)Click here for additional data file.S7 Fignekl-2(fd91) (B), nekl-3(sv3) (C) and nekl-2(fd81); nekl-3(gk894345) (D) animals. Wild type animals shown in A are the same animals from the main figure (A-D) Images of phalloidin staining in wild type (A), (TIF)Click here for additional data file.S8 Figcdc-42(gk388) larvae with molting defects. Wild type animals shown in A and B are the same animals from the main figure (A-F) Phalloidin staining of (TIF)Click here for additional data file.S9 Figx axis represents fluorescence intensity (gray level) in arbitrary units; y axis represents distance from the starting point of line scan in micrometers. Each line scan starts at the upper left corner; direction of each line scan is indicated by the arrowhead. Bar size in C = 10 μm in A–C.(A-C) Line scans of selected green (NeonGreen and GFP) and red (mCherry) puncta in representative images from (TIF)Click here for additional data file.S10 Fig2 at the apical surface of hyp7 measured in ten randomly selected animals for each indicated genomic background and feeding condition. Error bars represent standard deviations. p values were derived using a student’s t-test.Graphic representation of average number of GFP::CHC-1 puncta per 100 μm(TIF)Click here for additional data file.S11 Fignekl-2(fd81); nekl-3(gk894345); cdc-42(RNAi) animal that was superficially suppressed for molting defects but showed gross mislocalization of GFP::CHC-1. (C) Partial mislocalization of GFP::CHC-1 in a suppressed nekl-2(fd81); nekl-3(gk894345); sid-3(fd139) triple mutant. Color scale is shown in Wild type animal shown in A is the same as in the (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Tablesid-3(fd213), sid-3(fd214), sid-3(fd218), sid-3(fd219), and sid-3(fd221) mutants. Mutated regions are flanked upstream and downstream with ten wild-type nucleotides. Deleted regions are marked in red; insertions are indicated in blue. The number of the first nucleotide in each sequence, based on sid-3 isoform a, is shown in parentheses.Alignments of wild-type sequence and corresponding mutated regions in (DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


HRT and RPM1 levels and thereby pathogen resistance is significantly reduced in the cop1 mutant background. Notably, the levels of at least two double-stranded RNA binding (DRB) proteins DRB1 and DRB4 are reduced in the cop1 mutant background suggesting that COP1 affects HRT stability via its effect on the DRB proteins. Indeed, a mutation in either drb1 or drb4 resulted in degradation of HRT. In contrast to COP1, a multi-subunit E3 ligase encoded by anaphase-promoting complex (APC) 10 negatively regulates DRB4 and TCV resistance but had no effect on DRB1 levels. We propose that COP1-mediated positive regulation of HRT is dependent on a balance between COP1 and negative regulators that target DRB1 and DRB4.The E3 ubiquitin ligase COP1 (Constitutive Photomorphogenesis 1) is a well known component of the light-mediated plant development that acts as a repressor of photomorphogenesis. Here we show that COP1 positively regulates defense against turnip crinkle virus (TCV) and  Plants must constantly regulate the allocation of resources between photomorphogenesis and defense signaling. Although light is known to influence plant defense, the underlying mechanisms remain largely unknown. Here we show that light plays specific and direct signaling roles in plant defense. Specifically, a positive role for COP1 E3 ligase, an important regulator of photomorphogenesis, in plant defense is demonstrated. We further show that COP1 regulates the levels of double-stranded RNA binding proteins DRB1 and DRB4, which in turn regulate the levels of resistance protein HRT that confers resistance against turnip crinkle virus. In contrast to COP1, a multi-subunit E3 ligase encoded by anaphase-promoting complex (APC) 10 negatively regulates DRB4 but had no effect on DRB1 levels. Together, these results suggest that COP1-mediated positive regulation of HRT is dependent on a balance between COP1 and negative regulators that target DRB1 and DRB4. R) protein-mediated immunity is induced when a strain-specific avirulence (avr) protein from the pathogen associates with a cognate plant R protein [Resistance ( protein . Inducti protein . HR is o protein –5.Turnip crinkle virus (TCV) is activated by TCV coat protein (CP) [drb4 supports increased replication of TCV on the inoculated leaves of HRT containing plants and systemic spread to uninoculated parts [HRT drb4 or other HRT containing susceptible genotypes  do not accumulate viral specific small RNAs regardless of TCV levels in their inoculated leaves [Viruses have evolved to express suppressors that target host RNA silencing components and thereby ensure replication in the host –5. Interein (CP) , 7, whicein (CP) , 9. Howeein (CP) . Conversed parts . Notablyd leaves . This surrt (regulates resistance to TCV) [Compromised for Recognition of TCV; [trans-acting siRNAs (tasiRNAs), respectively [HRT is a coiled coil (CC)- nucleotide binding site (NBS)- leucine rich repeat (LRR) type R protein that is activated in the presence of CP , 11, 12, to TCV) . Resista to TCV) , 15. Amo to TCV) . HRT is  of TCV; , 17) but of TCV; . Interes of TCV; , and migectively , 19. DRBectively  and DRB3ectively .HRT-mediated resistance signaling is also dependent on blue-light photoreceptors , 21 and Here, we examined the role of COP1 in HRT-mediated resistance signaling. Surprisingly, we find that COP1 positively regulates HRT levels, and those of DRB1 and DRB4. Consistent with these results both DRB1 and DRB4 are required for resistance against TCV. In contrast to COP1, a multi-subunit E3 ligase encoded by anaphase-promoting complex (APC) 10 negatively regulates DRB4 and TCV resistance but had no effect on DRB1 levels. Our results suggest that COP1-mediated positive regulation of HRT is dependent on a balance between COP1 and negative regulators that target DRB1 and DRB4.cop1-6 plants  with Di-17 . Similar to Di-17 plants, the F2 progeny from the Di-17 x cop1 cross containing at least one copy of HRT and wild-type allele of COP1 developed visible and microscopic HR following TCV infection  of F2 progeny from a Di-17 x Col-0 cross showed typical crinkled leaves and drooping bolt phenotypes associated with susceptible plants. Only 25%  of these HR-developing progeny were able to resist TCV infection and did not allow the virus to spread into uninoculated tissues. In contrast, all HRT cop1 progeny showed susceptible phenotype suggesting that COP1 positively regulated HRT-mediated resistance to TCV  [cop1 with Col-0 plants expressing RPM1-Myc under its native promoter and generated cop1 RPM1-Myc plants. Interestingly, RPM-Myc protein levels were significantly reduced in the cop1 mutant background (RPM1 transcript levels in the cop1 background were comparable to those in COP1 plants (cop1 plants showed increased susceptibility to avrRpm1 Pst (Nicotiana benthamiana and co-immunoprecipitation (IP) assays in N. benthamiana and Arabidopsis. RPM1 did interact with COP1 and this interaction was primarily observed in the cytoplasm  . To thisckground , even th1 plants . This suRpm1 Pst . Next, wytoplasm . The BiFthamiana  and confthamiana . Togethecop1 plants contained reduced levels of both DRB1 and DRB4  phenotype (drb plants (Col-0 background) with Di-17 (TCV resistant ecotype). The F2 progeny from a Di-17 x Col-0 control cross or HRT introgressed into Col-0 background (backcrossed 8 times) were used as controls and both these genotypes developed visible and microscopic HR following TCV infection  of F2 progeny from a Di-17 x Col-0 cross showed typical crinkled leaf and drooping bolt phenotypes associated with TCV susceptibility. Only 25%  of these HR-developing progeny were able to resist TCV infection and did not exhibit virus spread to uninoculated tissues. Evaluation of genetic segregation in Di-17 x drb2 and Di-17 x drb3 crosses showed statistically significant deviation from Mendelian segregation; all HRT drb2 and HRT drb3 plants showed typical susceptible symptoms suggesting that DRB2 and DRB3 proteins were required for resistance to TCV .Next, we evaluated the segregation of resistant plants in Di-17 x e to TCV . The invb5 cross . Nonethee to TCV . Likewist plants . The reqHRT drb4, HRT crt1, and HRT cry2 genotypes, all of which showed reduced levels of HRT [HRT drb genotypes at 10 days post inoculation (dpi) showed pronounced chlorosis on HRT drb2, HRT drb3 and HRT drb5 but not HRT drb1 leaves , which contained the highest levels of TCV in inoculated leaves, did not show spreading lesions/cell death , increased the cytosolic pool of DRB4 and inhibited the HRT-DRB4 interaction [HRT drb1 plants. However, HRT drb4 showed normal HR at 3 dpi even though CP also inhibited the HRT-DRB4 interaction [HRT drb1 and DRB1 knock-down plants, suggested that DRB1 may be a dominant player in HR formation and thereby activation of HRT. Congruent with this notion, HRT cop1 plants that lack both DRB1 and DRB4 proteins showed loss of both visible as well as microscopic HR (Earlier we showed that TCV infection (in Arabidopsis), or CP expression  interacted with DRB4 and elevated levels of DRB4 in APC10 RNAi plants suggested that APC10 targeted DRB4 for degradation [APC10 overexpressing Col-0 plants [APC10 overexpressing plants showed reduced levels of DRB4, suggesting that the increased expression of APC10 negatively regulated accumulation of DRB4  DRB2 regulates HRT levels via its presence in HRT complexes comprising other DRB proteins. In fact, DRB2 and DRB4 were shown to interact with DRB1 and DRB5 in far-western assays [in planta, it is possible that they exist but are undetectable under the harsh conditions used for in planta IP assays. Weak interactions between DRB2 and DRB1/5 could enable the formation of multi-protein complexes that stabilize HRT. Loss of one or more components could disrupt such complexes resulting in the degradation of HRT. 2) DRB2 regulates HRT levels via its presence in HRT complexes comprising other proteins . Plants were grown on autoclaved Pro-Mix soil . Soil was fertilized once using Scotts Peter’s 20:10:20 peat lite special general fertilizer that contained 8.1% ammoniacal nitrogen and 11.9% nitrate nitrogen (Scottspro.com). Plants were irrigated using deionized or tap water.Plants were grown in MTPS 144 Conviron  walk-in-chambers at 22°C, 65% relative humidity and 14 hour photoperiod. The photon flux density of the day period was 106.9 μmoles mHRT line was generated after eight backcrosses of F1 derived from a Di-17 x Col-0 cross with Col-0, which was used as a recurrent parent. The F1 and F2 progenies from each backcross were genotyped for HRT and those from initial and final backcrosses were tested for HR and resistance phenotypes.Crosses were performed by emasculating the flowers of the recipient genotype and pollinating with the pollen from the donor. F2 plants showing the wt genotype at the mutant locus were used as controls in all experiments. The wt and mutant alleles were identified by PCR, CAPS, or dCAPS analysis. The Col-0-DRBs, the cDNA spanning the coding region were cloned into the pGWB5 vector [DRBs, the Myc or Flag-HA tagged DRBs along with their respective promoters were cloned into pCambia 1300 derived vector and transformed into respective drb mutant backgrounds. Genetic complementation was assayed by analyzing the levels of siRNA, as described before [The Di-17 and Col-0 transgenic plants expressing HRT-Flag transgene are described earlier . For tra5 vector , and expd before .PR-1, CP and DRB4 were carried out as described previously [Small-scale extraction of RNA from two or three leaves (per sample) was performed with the TRIzol reagent , following the manufacturer’s instructions. RNA gel blot analysis and synthesis of random-primed probes for eviously .260. Reverse transcription (RT) and first strand cDNA synthesis were carried out using Superscript II . Quantitative RT-PCR was carried out as described before [ACTIN II (At3g18780) expression levels were used as internal control for normalization. Cycle threshold values were calculated by SDS 2.3 software.RNA quality and concentration were determined by gel electrophoresis and determination of Ad before . Each saThe leaves were vacuum-infiltrated with trypan-blue stain prepared in 10 mL acidic phenol, 10 mL glycerol, and 20 mL sterile water with 10 mg of trypan blue. The samples were placed in a heated water bath (90°C) for 2 min and incubated at room temperature for 2–12 h. The samples were destained using chloral hydrate , mounted on slides and observed for cell death with a compound microscope. The samples were photographed using an AxioCam camera  and images were analyzed using Openlab 3.5.2 (Improvision) software.in vitro from a cloned cDNA of TCV using T7 RNA polymerase were used for viral infections. For inoculations, the viral transcript was suspended at a concentration of 0.05 μg/ μL in inoculation buffer, and the inoculation was performed as described earlier [Transcripts synthesized  earlier . After vavrRpm1 were grown overnight in King’s B medium containing rifampicin and kanamycin . The bacterial cells were harvested, washed and suspended in 10 mM MgCl2. The cells were diluted to a final density of 105 or 106 CFU/mL (A600) and used for infiltration. The bacterial suspension was injected into the abaxial surface of the leaf using a needle-less syringe. Three leaf discs from the inoculated leaves were collected at 0 and 3 or 6 dpi. The leaf discs were homogenized in 10 mM MgCl2, diluted 103 or 104 fold and plated on King’s B medium.The bacterial strain pVSP61 (empty vector), or 2, 5 mM EDTA, 5 mM DTT, and 1 X protease inhibitor cocktail . Protein concentration was measured by the Bio-RAD protein assay . For small scale extractions 2–3 leaves were homogenized per sample.Proteins were extracted in buffer containing 50 mM Tris-HCl, pH7.5, 10% glycerol, 150 mM NaCl, 10 mM MgClFor Ponceau-S staining, PVDF membranes were incubated in Ponceau-S solution (40% methanol (v/v), 15% acetic acid (v/v), 0.25% Ponceau-S). The membranes were destained using deionized water.Proteins (30–50 μg) were fractionated on a 7–10% SDS-PAGE gel and subjected to immunoblot analysis using α-CP, α-Myc, α-Flag  or α-GFP antibody. Immunoblots were developed using ECL detection kit (Roche) or alkaline-phosphatase-based color detection. Fold change, normalized with Rubisco, Actin or H3 proteins, in western blots was quantified using Image Quant software.Coimmunoprecipitations were carried out as described earlier , 15. BriNuclear fractionation was carried out as described before .For gel filtration experiments, ground mixed flower tissues were dissolved in lysis buffer . Supernatant was filtered through 0.45 μm membrane, and further processed by a 2 hours centrifugation, 4500 rpm on Amicon Ultra centrifugal units (Millipore). 500μl of the resulting crude extract was loaded onto the Superose 6 10/200 column  to perform size exclusion chromatography, 500μl/minute, and 500μl fractions were collected, precipitated separately in 2 volumes of absolute Ethanol overnight at 4°C, and pellets were resuspended in 100ul 2X Laemmli buffer. Separation and blotting was then performed as described above. Size markers were run in similar settings, in a separate run.A. tumefaciens strain LBA4404 for agroinfiltration into N. benthamiana and MP90 for Arabidopsis transformation. Agrobacterium strains carrying various constructs were infiltrated into wild-type or transgenic N. benthamiana plants expressing CFP-tagged nuclear protein H2B. 48 h later, water-mounted sections of leaf tissue were examined by confocal microscopy using a water immersion PLAPO60XWLSM 2 (NA 1.0) objective on a FV1000 point-scanning/point-detection laser scanning confocal 3 microscope (Olympus) equipped with lasers spanning the spectral range of 405–633 nm. RFP, CFP and GFP/YFP overlay images (40X magnification) were acquired at a scan rate of 10 ms/pixel. Images were acquired sequentially when multiple fluorophores were used. Olympus FLUOVIEW 1.5 was used to control the microscope, image acquisition and the export of TIFF files.For confocal imaging, samples were scanned on an Olympus FV1000 microscope . GFP (YFP), and RFP were excited using 488, and 543 nm laser lines, respectively. Constructs were made using pSITE , pEarlyGS1 FigRPM1 transcript in wild-type (Col-0) and cop1 mutant plants. This experiment was repeated twice using two or more independent cDNA preparations as templates. (B) Confocal micrographs showing BiFC for RPM1 and COP1. Agroinfiltration was used to express protein in transgenic N. benthamiana plants expressing the nuclear marker CFP-H2B . Arrows indicate nucleus. All interactions were confirmed using both combinations of reciprocal N-EYFP/C-EYFP fusion proteins in three separate experiments (three replicates per experiment). (C) Western blots showing relative levels of DRB2 in flowers from indicated genotypes. Ponceau-S staining of the Western blots was used as the loading control. Arrows indicate the target protein corresponding to the indicated antibody. This experiment was repeated three times with similar results.(A) Quantitative RT-PCR analysis showing relative levels of (TIF)Click here for additional data file.S2 FigA) Typical morphological phenotypes of four-week-old soil grown drb mutants plants. (B) Typical morphological phenotypes of TCV inoculated Di-17, HRT drb and Col-0 plants. Plants were photographed at 18 dpi.((TIF)Click here for additional data file.S3 FigA) Western blot showing relative HRT levels in F2 plants derived from Col-0-HRT x drb cross segregating for DRB1 (left panel) or DRB2 (right panel). Ponceau-S staining of the western blot was used as the loading control. This experiment was repeated with multiple F2 plants with similar results. (B) Quantitative RT-PCR analysis showing relative levels of HRT-FLAG transcript in drb mutant background. This experiment was repeated twice using two or more independent cDNA preparations as templates. (C) Typical morphological phenotypes of hrt drb and Col-0-HRT plants inoculated with TCV. Leaves were photographed at 10 dpi. (D) Confocal micrographs showing bi-molecular fluorescence complementation (BiFC) for indicated proteins. Agroinfiltration was used to express protein in transgenic N. benthamiana plants expressing the nuclear marker CFP-H2B . Arrow indicates nucleus. All interactions were confirmed using both combinations of reciprocal N-EYFP/C-EYFP fusion proteins in three separate experiments (three replicates per experiment). (E-H) Co-immunoprecipitation (IP) of DRB1-Myc (E), DRB2-Myc (F), DRB3-Myc (G) and DRB5-Myc (H) with HRT-Flag. N. benthamiana plants were agroinfiltrated and immunoprecipitated proteins were analyzed with α-Myc and α-Flag. HRT and DRB proteins were expressed under 35S promoter. This experiment was repeated twice with similar results. (I) Electrolyte leakage in N. benthamiana leaves infiltrated with buffer , or Agrobacterium cultures (suspended in the same buffer) expressing HRT, HRT+CP, HRT+CP+DRB or HRT+CP+EDS1. Error bars represent SD (n = 6). (J) Quantitative RT-PCR analysis showing relative levels of DRB transcripts in transgenic Di-17 plants overexpressing DRB1, DRB3 or DRB4. This experiment was repeated twice using two independent cDNA preparations as templates. Error bars indicate SD. Asterisks indicate data statistically significant from that of control (Di-17) . (K) HR formation in TCV-inoculated Di-17 and transgenic plants showing reduced- (line#1) or over-expression (line#6) of DRB1. The HR phenotype was evaluated in ~20 plants that were analyzed in two separate experiments. (L) Typical morphological phenotypes of TCV inoculated Di-17, Col-0 and transgenic plant showing reduced- (#1) or over-expression (#6) of DRB1. Plants were photographed at 18 dpi. (M) Percentage of resistant plants observed among indicated genotypes. The susceptible plants were scored based on crinkled and short bolt phenotypes. (N) Western blot showing relative levels of CP in the systemic bolt tissues of Di-17, Col-0 and three independent 35S-DRB1-1 plants. The plants were sampled at 14 dpi. Ponceau-S staining of the western blot was used as the loading control. This experiment was repeated three times with similar results. (O & P) Typical morphological phenotypes of Di-17 and 35S-DRB plants inoculated with a virulent R8A strain of TCV. Leaves (O) and plants (P) were photographed at 3 and 18 dpi, respectively. (Q) Western blot showing relative levels of TCV CP in Di-17 and 35S-DRB plants generated in Di-17 background. The inoculated leaves were sampled at 3 dpi. Ponceau-S staining of the western blot was used as the loading control. This experiment was repeated two times with similar results.((TIF)Click here for additional data file.S4 FigA) Typical growth phenotypes seen in wild-type (Col-0), cop1, and drb mutants grown under light (upper panel) or dark (lower panel) conditions. (B) Hypocotyl length in Col-0, cop1, and drb mutants grown under dark condition. These experiments were repeated twice with similar results.((TIF)Click here for additional data file.S5 FigA and B) Confocal micrographs showing localization of CP-RFP and DRB-GFP expressed individually (A) or coexpressed (B) in N. benthamiana. Arrows and arrowheads indicate nucleus and inclusion structures, respectively . This experiment was repeated three times (three replicates per experiment) with similar results. (C-F) Co-IP of DRB1-Flag (C), DRB2-Flag (D) and DRB3-Flag (E) and DRB5-Flag (F) with CP. N. benthamiana plants were agroinfiltrated and immunoprecipitated proteins were analyzed with α-CP and α-Flag. HRT and DRB proteins were expressed under 35S promoter. This experiment was repeated twice with similar results. (G-H) Yeast-two hybrid assay showing interaction between CP and DBR4. G shows growth on selection medium and H shows β-glactosidase assay. Yeast colonies co-expressing bait (pGADT7) and prey (pGBKT7) plasmids were streaked on plates without (-) leucine and tryptophan or without leucine, tryptophan, and histidine. (I) Confocal micrographs showing BiFC for indicated proteins. Agroinfiltration was used to express protein in transgenic N. benthamiana plants expressing the nuclear marker CFP-H2B . Arrows and arrowheads indicate nucleus and inclusion structures, respectively. All interactions were confirmed using both combinations of reciprocal N-EYFP/C-EYFP fusion proteins in three separate experiments (three replicates per experiment). (J) Confocal micrographs showing localization of indicated proteins. Agroinfiltration was used to express protein in transgenic N. benthamiana plants expressing the nuclear marker CFP-H2B . Arrows indicate nucleus. (K) Co-IP of DRB3-Myc or DRB4-Myc with HRT-Flag in the presence or absence of CP. N. benthamiana plants were agroinfiltrated and immunoprecipitated proteins were analyzed with α-Myc and α-Flag. This experiment was repeated two times with similar results.((TIF)Click here for additional data file.S6 FigA) Confocal micrographs showing BiFC for indicated proteins. Agroinfiltration was used to express protein in transgenic N. benthamiana plants expressing the nuclear marker CFP-H2B . Arrows indicate nucleus. All interactions were confirmed using both combinations of reciprocal N-EYFP/C-EYFP fusion proteins in three separate experiments (three replicates per experiment). (B) Hypocotyl length in Col-0, cop1, and APC10 overexpressing (OE) plants grown under dark condition. These experiments were repeated twice with similar results. (C) Model showing proteins interacting with COP1, APC10 and DRB4. This model includes both predicted and confirmed interactions and was created using Arabidopsis Interaction viewer. (D) Typical morphological phenotypes of soil grown four-week-old Col-0, cop1 and drb1 drb4 plants.((TIF)Click here for additional data file.S7 FigA) Model showing relationship between DRB interactomes. The model includes both predicted and confirmed interactions and was created using Arabidopsis Interaction viewer. The program did not predict any interactions for DRB3. (B) Distribution profile of DRB4-Myc and DRB2-FlagHA proteins, after size exclusion chromatography on a Superose 6 column. Both are detected in high molecular weight complexes, superior to 669 kDa. Five hundred microliter fractions were collected, precipitated and equivalent amounts were analyzed by western blot. Four separate gels were used to analyze one profile. Signals were detected with Myc and HA antibodies.((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


K422R) showed no enhancement role in iPSCs induction. Therefore, our data demonstrated that Pkm2 enhances the pluripotency maintenance in ESCs and promotes the pluripotency acquisition during somatic cell reprogramming.Aerobic glycolysis is one of the most important common characteristics in both cancer cells and stem cells. Metabolism switch has been discovered as an important early event in the process of reprogramming somatic cells to induced pluripotent stem cells (iPSCs). As a rate limiting kinase in glycolysis, Pkm2 has been reported playing critical roles in many tumors, yet its role in stem cells and iPSCs induction is poorly defined. In the present study, we showed that Pkm2 is a predominant pyruvate kinase in embryonic stem cells (ESCs), and its expression increases many pluripotent genes. During somatic cell reprogramming, up-regulation of Pkm2 can be observed and over-expression of Pkm2 can facilitate iPSCs induction, while Pkm1 or a mutant form of Pkm2 (Pkm2 By ectopically expressing Oct4, Sox2, Klf4 and c-Myc, somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) . ExploriAerobic glycolysis has been extensively studied in many tumors. Cancer cells maintained highly glycolysis activity even in the aerobic environment called Warburg effect, thus to generate ATP faster and provide more building blocks to meet the anabolic demands of higher proliferation . AccordiPkm2 was known to abundant in proliferating cells, embryonic tissues, and stem cells besides tumor cells . But itsIt has been shown previously that Pkm2 was highly expressed in embryonic tissue and cancer cells . Here, w*M2)Jaetm1(rtTA (rtTA) and Tg(Pou5f1-EGFP)2Mnn (OG2) mice. Doxycycline (Dox) controlled inducible lentiviral overexpression vectors carrying Pkm1, Pkm2 or empty vector tagged with fluorescence protein monomeric Kusabira orange (mKO) was introduced into the rtTA-OG2 ESCs in which EGFP signal indicating the expression of endogenous Oct4. We established stable cell lines with normal colony appearance and Oct4-EGFP positive signal 26Sorl Figure , which el Figure  and 2B. l Figure . Then wel Figure . Thus, oAlthough Pkm2 could increase the expression of some pluripotent genes, the role of it in pluripotent cells seems not so crucial, as the availability of KO mice of Pkm2 has been reported previously . We empl*M2)Jaetm1Jae (rtTA-OSKM) and Tg(Pou5f1-EGFP)2Mnn (OG2) to establish a secondary reprogramming system [Based on the role of Pkm2 in pluripotency maintenance, we next aimed to investigate its function in pluripotency acquisition during somatic cell reprogramming. We employed the transgenic mice: Gt(ROSA)26Sorg system . The somK422R even impaired iPSC induction efficiency Jaetm1Jae and Tg(Pou5f1-EGFP)2Mnn transgenic mice were employed in this study.Gt(ROSA)26SorSpecific pathogen-free mice were housed in the animal facility of Dalian Medical University. All studies adhered to procedures that were consistent with the Dalian Medical University Guide for the care and use of laboratory animals.ESCs and iPSCs were cultured on mitomycin C treated MEFs in ESC culture medium composed with DMEM (Merk Millipore) supplemented with 15% (v/v) fetal bovine serum (Hyclone), 1 mM L-glutamine (Merk Millipore), 0.1 mM mercaptoethanol (Invitrogen), 1% nonessential amino acid stock (Merk Millipore), nucleosides  and 1000 U/ml LIF (Merk Millipore).*M2)Jaetm1Jae crossed with Tg(Pou5f1-EGFP)2Mnn transgenic mice. After culturing in ESC culture medium containing Dox for 12 days, ESC-like colonies appeared, and then the Dox was removed from the culture medium. ESC-like colonies were individually digested and replated. After propagation, we selected iPS cell lines that exhibited typical ES cell morphology for long-term culture.For iPSCs induction, MEFs were derived from Gt(ROSA)26SorESCs were seeded in the culture plate with Ultra-low attachment surface (Corning) in differentiation medium composed with DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (Hyclone), 1 mM L-glutamine (Merk Millipore), 0.1 mM mercaptoethanol (Invitrogen), 1% nonessential amino acid stock (Merk Millipore), nucleosides . After 5 days, the EBs were transferred to the gelatin coated cell culture dish for further differentiation.Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed by 5×All-In-One RT MasterMix (Abm). Real-time PCR was performed with KAPA SYBR fast qPCR kit on the Real-time PCR System (Applied Biosystems). β-Actin served as internal control.Pkm1-F: GCCTCCAGTCACTCCACAGA, Pkm1-R: CAGCACGGCATCCTTACACA; Pkm2-F: CAGCACCT GATTGCCCGAGA, Pkm2-R: CCAGACTTGGTGAGCAC GATA; Pklr-F: CTATGGCGGACACCTTCC, Pklr-R: TGT TCATCCCTGCCTTGAT; Nanog-F: CACCCACCCATG CTAGTCTT, Nanog-R: ACCCTCAAACTCCTGGTCCT; Eras-F: ACTGCCCCTCATCAGACTGCTACT, Eras-R: CA CTGCCTTGTACTCGGGTAGCTG; Rex1-F: ACGAGTGG CAGTTTCTTCTTGGGA, Rex1-R: TATGACTCACTTCC AGGGGGCACT; Oct4-F: CTGAGGGCCAGGCAGGA GCACGAG, Oct4-R: CTGTAGGGAGGGCTTCGGG CACTT; Fgf5-F: AACTCCATGCAAGTGCCAAAT, Fgf5 -R: CGGACGCATAGGTATTATAGCTG; Afp-F: CTTC CCTCATCCTCCTGCTAC, Afp-R: ACAAACTGGGTAAA GGTGATGG; Gata6-F: TTGCTCCGGTAACAGCAGTG, Gata6-R: GTGGTCGCTTGTGTAGAAGGA; Actin-F: AGAGGGAAATCGTGCGTGAC, Actin-R: CAATAG TGATGACCTGGCCGT.The cells were harvested, washed with cold PBS, and lysed with RIPA buffer. The lysates were centrifuged at 16,000 g for 10 minutes at 4°C to remove cell debris. Proteins was analyzed under denaturing conditions in 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes. Blots were blocked in TBST buffer supplemented with 5% skim milk for 1 hour, followed by probing with primary antibodies 4°C overnight. After three washes with TBST buffer, the blots were incubated with secondary antibodies conjugated to HRP in TBST buffer for 1 hour, followed by three washes with TBST buffer. ECL Prime Western Blotting detection reagent  was used to generate chemiluminescence signals which were detected by Chemi Doc Touch imaging system (Bio-Rad). ß-ACTIN was used as a loading control.The antibody against PKM2 was from Cell Signaling Technology. PKM1 antibody was from Proteintech. ß-ACTIN anbibody was from Transgen Biotech.Alkaline Phosphatase staining was performed according to the manufacturer's recommendation using the Alkaline Phosphatase Detection Kit (Millipore).* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.Each experiment was performed with a minimal of three biological replicates and the replicate number is given in the figure legends. Mean and standard deviation were calculated as indicated. Statistical analyses used unpaired, Student's t tests to test significance. Where indicated, ns = not significant,


A wealth of evidence indicates that inflammation plays a central role in the current paradigm of liver fibrosis. Kupffer cells, which represent the largest population of resident macrophages in the body , are uni4)-induced cirrhosis [Arachidonic acid metabolites derived from 5-lipoxygenase (5-LO) are essential regulators of cell growth and survival . Given t4 as described elsewhere [Liver injury was induced in male adult Wistar rats by inhalation of CCllsewhere .in situ collagenase perfusion and purified by Percollà density gradients as previously described [Liver cells were isolated by escribed ,7. Kupffescribed .4 synthase and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cDNA sequences. PCR products were analyzed by gel electrophoresis.Total RNA was obtained by the guanidinium isothiocyanate-cesium chloride method. RT was performed using an avian myeloblastoma virus reverse transcriptase cDNA synthesis kit. PCR was performed using oligonucleotides designed from published rat 5-LO, 5-LO-activating protein (FLAP), LTC4 and LTC4/LTD4/LTE4 (cysteinyl-LT) levels were quantified in cell supernatans of freshly isolated rat Kupffer cells (1à2.8 à 106 cells) maintained in culture for 16 hours by specific EIA kits. 5-hydroxyeicosatetraenoic acid (5-HETE) was analyzed by RP-HPLC.LTB6 cells) were cultured for up to 6 days in complete RPMI 1640 medium and cell growth was determined by the microculture MTT assay. To ascertain the effects of 5-LO inhibitors on cell survival, Kupffer cells from cirrhotic livers were grown in the presence of vehicle, AA861 (10 micromolar) and BAY-X-1005 (30 micromolar) for 8 h at 37 degrees C and the number of cells examined by direct counting using the Neuebauer chamber. The effects of 5-LO inhibitors on cell proliferation were further assessed in THP-1 cells by the MTT assay.Rat Kupffer cells  or BAY-X-1005 (30 micromolar) for 96 hours at 37 degrees C. DNA fragmentation was detected using the TACSà DNA Laddering kit and visualized by agarose gel electrophoresis.Nuclear morphology was assessed by optical microscopy visualization in Diff-QuikTHP-1 cells were incubated in the presence of vehicle, AA861 (10 micromolar) or BAY-X-1005 (30 micromolar) at 37 degrees C. After 72 h, cells were stained with propidium iodide and DNA content frequency cell cycle distribution analyzed by means of fluorescence-activated cell sorting (FACS) analysis.4 and cysteinyl-LT [Among the different hepatic sinusoidal cell types, Kupffer cells have been historically considered to possess the capacity to produce most of liver arachidonic acid metabolites including the 5-LO products LTBeinyl-LT . Indeed,4 and cysteinyl-LT  and by the FLAP inhibitor BAY-X-1005 (55.2 à 2.5% inhibition). These findings were further characterized in THP-1 cells where AA861 and BAY-X-1005 inhibited proliferation in a dose- and time-dependent fashion. In these cells, the antiproliferative effect was associated with induction of programmed cell death, as evaluated by using different techniques for apoptosis detection (see "Methods").4 and cysteinyl-LT. Cysteinyl-LT are also produced in hepatocytes by transcellular metabolism of LTA4 formed by Kupffer cells [The current knowledge of the 5-LO pathway in liver sinusoidal cells is shown in figure