Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). MicroRNAs (miRNAs) are involved in host-pathogen interactions and innate immune response to viral infection. However, the role of miRNAs in host response to IBDV infection is not clear. We report here that gga-miR-155 acts as an anti-virus host factor inhibiting IBDV replication. We found that transfection of DF-1 cells with gga-miR-155 suppressed IBDV replication, while blockage of the endogenous gga-miR-155 by inhibitors enhanced IBDV replication. Furthermore, our data showed that gga-miR-155 enhanced the expression of type I interferon in DF-1 cells post IBDV infection. Importantly, we found that gga-miR-155 enhanced type I interferon expression via targeting SOCS1 and TANK, two negative regulators of type I IFN signaling. These results indicate that gga-miR-155 plays a critical role in cell response to IBDV infection. It has been reported that chicken type I interferon has strong antiviral activity in IBDV-infected cells .DF-1 cells  were obtained from ATCC. Cells were cultured in Dulbecco modified Eagle medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 incubator. PGL3-Control vector was kindly provided by Wenhai Feng . Restriction enzyme XbaI was purchased from TaKaRa, Poly(I:C) from Sigma, and HRP-conjugated goat anti-mouse IgG and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG from Ding-Guo (China). Anti-VP3 monoclonal antibody and anti-TANK polyclonal antibodies were developed in our laboratory. All miRNA mimics and inhibitors  were synthesized by GenePharma Company .GSE90095). miRNA targets in host cells were predicted by TargetScan  and miRanda .Deep sequencing was performed by LC Sciences  on DF-1 cells infected with mock or IBDV at an MOI of 1 for 24 h  were transfected using Lipofectamine 2000 transfection reagents (invitrogen). Twelve hours after transfection, cells were infected with IBDV. Twenty-four hours after IBDV infection, cell cultures were collected for virus titration or RNA quantification, or cells were fixed for indirect immunofluorescent antibody assay.5 cells/ml. Cells were cultured for 7 days at 37°C in 5% CO2. Tissue culture wells with a cytopathic effect (CPE) were determined as positive. The titer was calculated on the basis of a previously described method  in DF-1 cells. Briefly, the viral solution was diluted by 10-fold in DMEM. A 100 μl aliquot of each diluted samples was added to the wells of 96-well plates, followed by addition of 100 ul of DF-1 cells at a density of 5 × 10Lx strain at an MOI of 0.01 for 24 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 1% bovine serum albumin, and probed with mouse anti-VP3 monoclonal antibody, followed by incubation with FITC-conjugated goat anti-mouse antibody (green). Cells were examined with a fluorescence microscope .DF-1 cells were transfected with miRNA mimics or miRNA controls for 12 h at the concentration of 200 nM, and followed by infection with IBDV TM RT Reagent kit (Takara). Quantitative RT-PCR analysis was performed using Tli RnaseH Plus (Takara) on LightCycler 480II . Specific primers for chicken IFN-α (chIFN-α) (5′-CCA GCA CCT CGA GCA AT-3′ and 5′-GGC GCT GTA ATC GTT GTC T-3′), chicken IFN-β (chIFN-β) (5′-GCC TCC AGC TCC TTC AGA ATA CG-3′ and 5′-CTG GAT CTG GTT GAG GAG GCT GT-3′), chicken IRF3 (chIF3) (5′-GCT CTC TGA CTC TTT CAA CCT CTT-3′ and 5′-AAT GCT GCT CTT TTC TCC TCT G-3′), and chicken GAPDH (5′-TGC CAT CAC AGC CAC ACA GAA G-3′ and 5′-ACT TTC CCC ACA GCC TTA GCA G-3′) were designed with reference to previous publications , and a 0.4 μM of each gene-specific primers. Thermal cycling parameters were as follows: 94°C for 2 min; 40 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 20 s; and 1 cycle of 95°C for 30 s, 60°C for 30 s, and 95°C for 30 s. The final step was to obtain a melt curve for the PCR products to determine the specificity of the amplification. qRT-PCR analysis of gga-miR-155 was performed with RT-PCR Quantitation Kit .Total RNA and miRNA were prepared from DF-1 cells using EASYspin Plus kit or RNA mini kit  per the manufacturer's instructions. mRNAs were reversely transcribed with primescriptATTA A-3′ mutated to 5′-TCA GAT CTA AGT ACA GC TAAT A. The first seed sequence of TANK: 5′-TGC TAA TAT ACC TTT CCA GC ATTA ATCA-3′ mutated to 5′-TGC TAA TAT ACC TTT CCA GC TAAT ATCA-3′, and the second seed sequence of TANK: 5′-TGG TTT TTG ATA AAA TTA GC ATTA ATAT-3′ mutated to 5′-TGG TTT TTG ATA AAA TTA GC TAAT ATAT-3′) using a fast mutagenesis system  per the manufacturer's instructions. Another vector pRL-TK containing the Renilla luciferase was used as a control.A 349 bp fragment of SOCS1 gene or a 480 bp fragment of TANK gene around the predicted gga-miR-155 target sites were amplified and inserted into the site downstream of the firefly luciferase gene in the pGL3-Control vector at the XbaI site to create the wild type 3′UTR vector. Both miRNA seed sites mutant in SOCS1 gene or TANK gene were made by mutating the underlined 4 nucleotides in the 8mer seed sequences (SOCS1: 5′-TCA GAT CTA AGT ACA GC socs1 gene was cloned from DF-1 cells using the specific primes 5′- CGC GGA TCC ATG GTA GCG CAC AGC AAG GTG-3′ (sense), and 5′- ACG CGT CGA CG TTA GAT CTG AAA CGG GAA GGA-3′ (anti-sense) with reference to the sequence in GenBank (accession no.NM_001137648.1). Chicken socs1 gene was then subcloned into pET-28a vector. The pET-28a-socs1 recombinant plasmid was used to transform E. coli BL21, and SOCS1-his recombinant protein was expressed at 37°C and purified by a protein purification kit  per the manufacturer's instructions. To develop polyclonal antibody against SOCS1, BALB/c mice were immunized with SOCS1-his recombinant protein. After immunization, anti-SOCS1 polyclonal antibody was obtained from the sera of BALB/c mice vaccinated with SOCS1-his recombinant protein. For detection of TANK in DF-1 cells, cell lysates were prepared using a non-denaturing lysis buffer . The cell lysates were boiled with 6 × SDS loading buffer for 10 min and fractionated by electrophoresis on 12% SDS-polyacrylamide gels, and resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skimmed milk, the membranes were incubated with anti-TANK or anti-GAPDH antibody, followed by HRP-conjugated anti-Mouse secondary antibody. Blots were developed using an enhanced chemiluminesence (ECL) kit per the manufacturer's instruction.Chicken The siRNA was designed by GenePharma Company  and used to knockdown TANK in DF-1 cells. The siRNAs for targeting TANK in DF-1 cells included the followings: RNAi#1 , RNAi#2 , RNAi#3 , and negative control . DF-1 cells were seeded on 12-well or 24-well plates. Twenty-four hours after culture, cells were transfected with siRNA using RNAiMAX per the manufacturer's instructions (Invitrogen). Double transfections were performed at a 24 h interval. Twenty-four hours after the second transfection, cells were harvested for further analysis.The significance of the differences between gga-miR-155-transfected cells and controls in gene expression, and viral growth was determined by the Mann-Whitney test and analysis of variance (ANOVA) accordingly.Lx strain at an MOI of 1 for 24 h. Among the differentially expressed miRNAs, 369 miRNAs were significantly upregulated and 169 downregulated  induced upregulation of miR-155 in different cell lines prompted us to investigate the possibility that upregulation of gga-miR-155 in IBDV-infected DF-1 cells is related to type I IFN signaling . Similarly, gga-miR-155 inhibitor also markedly knocked down the expression of endogenous gga-miR-155 by about 13 folds relative to the inhibitor controls in cells with poly(I:C) treatment  and pGL3-3′UTR-WT (TANK) containing the predicted target site in the 3′UTR, and four mutant vectors pGL3-3′UTR-Mut (SOCS1), pGL3-3′UTR-Mut1 (TANK), pGL3-3′UTR-Mut2 (TANK) and pGL3-3′UTR-Mut1,2 (TANK) with mutations of four nucleotides in the seed region  and pGL3-3′UTR-WT (TANK), but not pGL3-3′UTR-Mut (SOCS1) or pGL3-3′UTR-Mut (TANK)   Figure . Further) Figure . It was ) Figure , and ourTo examine the role of TANK in suppression of viral replication by gga-miR-155, we made three TANK RNAi constructs, and we found that one could effectively lower the cellular level of TANK without causing discernible changes in cell morphology Figure . We thenTaken together, our data show that IBDV infection induces gga-miR-155 expression and that miR-155 enhances type I IFN signaling and suppresses IBDV replication by targeting SOCS1 and TANK, two negative regulators in the immune response Figure .50 assay, we found that only gga-miR-155 significantly suppressed IBDV replication.IBDV infection causes severe damages in lymphoid organs in birds, especially the bursa of Fabricius  with human SOCS1 (GenBank accession no. DQ086801.1) is only around 60.6%. Our data show that gga-miR-155 markedly suppressed SOCS1 expression. TANK plays versatile roles in innate immune signaling. On one hand, TANK serves as an adaptor bridging TRAF3 with TBK1 and IKKϵ, which promotes phosphorylation and activation of IRF3/IRF7 as well as induction of NF-κB activation, leading to efficient type I IFN production (Guo and Cheng, The interaction between hsa-miR-155 and SOCS1 has been reported in mammalian cells (Jiang et al., Of note, the mechanism underlying the interaction of host miRNAs and pathogenic infection is intricate. Host cells employ miRNAs to reduce pathogenic infection, while some pathogens encode miRNAs or manipulate host miRNAs expression to benefit their replication (Lu et al., In summary, our results reveal that IBDV infection induces gga-miR-155 expression. We found that miR-155 enhanced type I IFN signaling and suppressed IBDV replication by targeted SOCS1 and TANK, two negative regulators in the immune response. These findings have provided insights for further studies of the molecular mechanism underlying host response to IBDV infection.SZ and BW designed and conceived the experiments, conducted data analysis and wrote the paper. BW and MF performed the experiments. YL, YW, XL, and HC contributed reagents, materials and analysis tools.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AG and handling Editor declared their shared affiliation.


Erratum to: npj Microgravity (2016); doi: 10.1038/s41526-016-0001-9 (2017); Published online 19 December 2016In this Article, data within Supplementary Figure 1 inadvertently included information that breached subject confidentiality. This information has been removed from Supplementary Figure 1 in the online versions of the paper.


C. elegans germ line, through DAF-18/PTEN, inhibits adult germline stem cell (GSC) proliferation, despite high systemic IIS activation. We show here that this feedback occurs through a novel cryptic signalling pathway that requires PAR-4/LKB1, AAK-1/AMPK and PAR-5/14-3-3 to inhibit the activity of MPK-1/MAPK, antagonize IIS, and inhibit both GSC proliferation and the production of additional oocytes. Interestingly, our results imply that DAF-18/PTEN, through PAR-4/LKB1, can activate AAK-1/AMPK in the absence of apparent energy stress. As all components are conserved, similar signalling cascades may regulate stem cell activities in other organisms and be widely implicated in cancer.Under replete growth conditions, abundant nutrient uptake leads to the systemic activation of insulin/IGF-1 signalling (IIS) and the promotion of stem cell growth/proliferation. Activated IIS can stimulate the ERK/MAPK pathway, the activation of which also supports optimal stem cell proliferation in various systems. Stem cell proliferation rates can further be locally refined to meet the resident tissue’s need for differentiated progeny. We have recently shown that the accumulation of mature oocytes in the  Adult stem cell proliferation rates respond to the needs for their differentiated progeny. For example, the lung stem cells of a smoker will divide more frequently than those of a non-smoker to constantly replenish smoke-damaged tissues. Molecularly, stem cell proliferation rates vary according to growth factor levels, some of which, such as insulin, are regulated systemically. What remains unclear is whether and how a more or less rapid turnover of differentiated cells interferes with growth factor signalling to ultimately set the stem cells’ proliferative rate. We show here that this is accomplished though a novel cryptic signalling pathway that locally antagonizes the effects of insulin through inactivating a more locally-acting growth factor pathway . Interestingly, a ubiquitous protein kinase (AMPK) that has primarily been implicated in energy stress response constitutes an integral part of this feedback mechanism. This suggests that an abundance of differentiated cells is molecularly interpreted as being similar to energy stress at the level of the stem cells, likewise leading to the inhibition of their growth and proliferation. Stem cells can divide symmetrically to generate more copies of themselves or asymmetrically to produce one stem cell and a daughter cell destined for differentiation. Whether a stem cell will proliferate or differentiate is governed by niche signaling, which locally prevents differentiation . Stem ceC. elegans (Ce), activated IIS stimulates germline stem cell (GSC) proliferation throughout postembryonic life  (generously provided by Drs Dan Dickinson and Bob Goldstein . LGX: aak-2(ok524). The following rearrangements were used. qC1]III, hT2[bli-4(e937) let-?(q782) qIs48], mIn1 [mIs14 dpy-10(e128)] II, nT1[unc-?(n754) let-?] . RNAi was induced by feeding from the L1 stage as previously described -histone H3, to mark M-phase nuclei. Undifferentiated germ nuclei counting in 3 dimensions was partially automated as described [fog-1, fog-1; daf-18, oma-1; oma-2, daf-2; [GSC mitotic indexes were evaluated as previously described , by tranescribed , with an, daf-2; ), we didA1 animals were generated as above, fixed in Carnoy’s solution and stained with DAPI as previously described . The numWhole animals were fixed in Carnoy’s solution and stained with DAPI . For gerfog-2, A1).Images were acquired at a 0.75 μm Figs  and 2B oS1 FigA-E) Representative A1 animals of the indicated genotypes were stained with DAPI. A white dotted line underlines the region occupied by arrested unfertilized oocytes. A red dotted line marks the uterine region, occupied by (A) embryos, (C-E) spontaneously matured endomitotic oocytes, or (B) empty. Anterior is to the left and dorsal, is up. Alleles, fog-1(q185), daf-18(nr2037), par-4(it57), aak-1(tm1944), aak-2(ok524). Scale bar, 100 μM.((TIF)Click here for additional data file.S2 FigA-E) Representative A1 proximal gonads extruded from animals of the indicated genotypes, stained with DAPI (cyan) and anti-MEX-3 (green), are shown. Diakinesis oocytes are negatively labeled from the proximal-most one. Positive numbers indicate ovulated oocytes (in utero) and asterisks mark activated oocytes (endomitotic). Proximal is to the left. Alleles, fog-1(q185), daf-18(nr2037), par-4(it57), aak-1(tm1944), par-5(it55). Scale bar, 20 μM.((TIF)Click here for additional data file.S3 FigA) The GSC MIs of A1 hermaphrodites of the indicated genotypes/conditions were determined. An asterisk indicates statistical significance  to all other samples. n, 18–34. (B) The number of GSC per animal at the dauer stage was evaluated as previously described [A-B) Dots mark averages, open boxes and brackets represent the whole sample, divided in quartiles. ns, Not significant. Alleles, daf-2(e1370), par-4(it57), let-60(ga89)gf. qIs56 is present in the daf-2; par-4 strain.(escribed  in anima(TIF)Click here for additional data file.S4 FigD-O) The dpMPK-1 signal intensity in arbitrary units (A.U.) of fluorescence was background subtracted and plotted as a function of the distance across germline zone I (from early/mid-pachytene to oocytes). As fog-2 mutants having accumulated oocytes are negative for dpMPK-1 [Click here for additional data file.S5 FigD’-P”‘) Additional representative germ lines extruded from unmated or continually mated animals of the indicated genotypes and ages, stained with DAPI  and a monoclonal mouse anti-MAPKYT antibody labeling dpMPK-1 (green). (P’-P”‘) A1 fog-1 germ lines were indistinguishable from A1 fog-2 germ lines (E’-E”). Maximal projections in (J”) did not stitch properly.((TIF)Click here for additional data file.S6 FigA-B) Three representative A1 animals of the indicated genotypes stained with DAPI are shown. Germline tumours grow noticeably slower in the absence of mpk-1. Anterior is to the left and dorsal, is up. Scale bar, 50 μM. (C) The GSC MIs of A1 hermaphrodites of the indicated genotypes were determined. An area located around the bend of the germ line was randomly selected using the DAPI channel for MI analysis. ns, Not significant . n, 25–30. (A-C) Alleles, gld-3(q730), nos-3(q650), mpk-1(ga117).((TIF)Click here for additional data file.


Androgen receptor (AR) and PI3K/AKT/mTORC1 are major survival signals that drive prostate cancer to a lethal disease. Reciprocal activation of these oncogenic pathways from negative cross talks contributes to low/limited success of pathway-selective inhibitors in curbing prostate cancer progression. We report that the antibiotic salinomycin, a cancer stem cell blocker, is a dual-acting AR and mTORC1 inhibitor, inhibiting PTEN-deficient castration-sensitive and castration-resistant prostate cancer in culture and xenograft tumors. AR expression, its transcriptional activity, and androgen biosynthesis regulating enzymes CYP17A1, HSD3β1 were reduced by sub-micro molar salinomycin. Estrogen receptor-α expression was unchanged. Loss of phosphorylated AR at serine-81, which is an index for nuclear AR activity, preceded total AR reduction. Rapamycin enhanced the AR protein level without altering phosphoAR-Ser81 and CYP17A1. Inactivation of mTORC1, evident from reduced phosphorylation of mTOR and downstream effectors, as well as AMPK activation led to robust autophagy induction. Apoptosis increased modestly, albeit significantly, by sub-micro molar salinomycin. Enhanced stimulatory TSC2 phosphorylation at Ser-1387 by AMPK, and reduced inhibitory TSC2 phosphorylation at Ser-939/Thr-1462 catalyzed by AKT augmented TSC2/TSC1 activity, which led to mTORC1 inhibition. AMPK-mediated raptor phosphorylation further reduced mTOR's kinase function and mTORC1 activity. Our novel finding on dual inhibition of AR and mTORC1 suggests that salinomycin is potentially active as monotherapy against advanced prostate cancer. Mechanistic Target of Rapamycin Complex-1 (mTORC1) activities are two key drivers of prostate cancer. Apoptosis of castration-sensitive prostate cancer cells in response to androgen deprivation therapy (ADT) causes initial regression of the disseminated disease, although recurrence to a castration-resistant lethal phenotype is inevitable, and for ~70% post-ADT patients cancer progresses within 20 months [Androgen receptor (AR) and 0 months , 4. Inhi0 months –8. Synth0 months .Ras Homolog Enriched in Brain), which is a positive regulator of mTORC1. The mTORC1 complex is a hub for signal convergence from numerous positive and negative regulatory cascades. TSC2/TSC1 inhibition due to AKT-mediated TSC2 phosphorylation at serine-939 and threonine-1462 leads to mTORC1 activation, whereas mTORC1 inhibition and consequent cell growth arrest in response to nutrient deprivation or oxidative stress is caused by activation of AMP-activated kinase (AMPK) and a phosphorylation-dependent cascade that ensues. TSC2 phosphorylation at serine-1387 by AMPK stimulates TSC2/TSC1 activity, thus inhibiting mTORC1. Raptor phosphorylation at serine-722 and Ser-792 by AMPK also inactivates mTORC1 [The PI3-kinase/AKT/mTORC1 oncogenic axis is activated in ~40% primary prostate cancer and ~70% mCRPC, ascertained on the basis of whole-genome exome profiling of tumor samples from several hundred patients . The serActivation of the PI3-kinase (PI3K) axis from PTEN deficiency suppressed AR transcriptional output in murine models of prostate cancer , 16. Howin vitro and inhibition of prostate tumor growth in vivo in xenograft tumor models. Loss of serine-81 AR phosphorylation preceded total AR reduction in salinomycin-treated cells. Inhibition of mTORC1 was associated with enhancement of AMPK-mediated phosphorylation of TSC2 and raptor, as well as reduction of TSC2 phosphorylation by AKT. Our results suggest that salinomycin may be clinically active as monotherapy against advanced prostate cancer.Herein we provide the first evidence that salinomycin, a potassium ionophore and a human cancer stem cell inhibitor, can lower AR expression and activity, reduce protein levels of CYP17A1 and HSD3β1, the critical regulators of the androgen biosynthesis pathway, and block mTORC1 activity in PTEN-deficient prostate cancer cells. These molecular changes are associated with reduced proliferation of castration-sensitive and castration-resistant prostate cancer cells Salinomycin inhibited cell proliferation for AR-expressing LNCaP (castration-sensitive) and C4-2B (castration-resistant) human prostate cancer cells Figure . InhibitAnnexinV+PI− cells, indicating early apoptosis, increased significantly after cells were treated with the drug for 3 days Figure . The modAutophagy induction by salinomycin was revealed from elevated LC3B levels in LNCaP-II (an LNCaP variant) and C4-2B cells Figure . Autophaversus salinomycin-treated RWPE-1 cells  and the downstream effector ribosomal protein S6 (RPS), which is an S6K substrate Figures . Marked s Figure . The enhs Figure  is consis Figure . P-RPS ds Figure . P-RPS ws Figure . P-RPS rs Figure . Other is Figure . Reductis Figure . Decreass Figure .Salinomycin reduced AR mRNA and protein levels in LNCaP and C4-2B cells Figures  & 4B. ReAR phosphorylation at serine-81, a metric of its nuclear activity, was inhibited by salinomycin -- the inhibition preceding total AR reduction Figure . In C4-2CYP17A1 (cytochrome P450 lyase/hydroxylase) and HSD3β1 (hydroxysteroid dehydrogenase-3β1) are key regulators of the androgen biosynthesis pathway and thus, the intratumoral androgen level. Reduced expression of both enzymes in C4-2 cells was observed upon 20hr incubation with salinomycin. No change in CYP17A1 and HSD3β1 was detected at 6 hour Figure . GAPDH . The mRN3. For LNCaP-II xenografts, intraperitoneal (i.p.) injections of salinomycin (5 mg/Kg body weight) or vehicle (DMSO) were administered every 3rd day till day-16, after which tumors were harvested. Tumor growth rate declined significantly for the salinomycin group after 6 injections compared to the vehicle control p<0.04, n=5  and prostate histology  levels were elevated by salinomycin in prostate cancer cells . Since oReduced TSC2 phosphorylation at Ser-939 and Thr-1462 (two AKT-targeted sites) within 6hr drug treatment Figure  also conin vitro and in tumor xenografts, and prevented a compensatory negative crosstalk between AR and mTORC1, which otherwise would have led to reciprocal activation of the two oncogenic pathways, causing activation of one when the other is blocked. This compensatory interplay is believed to have contributed to the low activity of a combined regimen of everolimus (mTORC1 inhibitor) and bicalutamide (AR antagonist) against metastatic prostate cancer in a phase II trial [The present study identified salinomycin as the first example of a dual-acting inhibitor of AR and mTORC1. Salinomycin, a polyether antibiotic, inhibited PTEN-null castration-sensitive and castration-resistant prostate cancer cells for expansion II trial . This clII trial , 17, 33.Figure Increased RPS phosphorylation by the synthetic androgen R1881 is consistent with previous reports that androgen stimulated mTORC1 in LNCaP cells due to induction of nutrient transporters, which would increase nutrient availability , 22. RobImpaired AR signaling by salinomycin due to interference with AR's Ser-81 phosphorylation, AR mRNA and protein reduction and inhibition of AR transcriptional readout – all of which are novel findings in the present study- contrasts rapamycin-mediated increase in the AR level and activity, which likely contributed to the clinical failure of everolimus against castration-resistant prostate cancer , 43. CYPERK1/2 Map kinase was activated by salinomycin and rapamycin , which iAMPK activation by salinomycin Figure  is in kein vitro and in xenograft models. The drug did not cause overt toxicity, since tumor growth inhibition was not associated with changes in prostate histology ; rapamycin, LC Laboratories ; DPT, T0901317 ; EB1089 . LNCaP, RWPE-1 and PC-3 are from American Type Culture Collection ; LNCaP-II, C4-2, C4-2B, ViroMed Laboratories Inc. . RWPE-1 cells were cultured in keratinocyte medium plus 5% fetal bovine serum (FBS), supplemented with penicillin and streptomycin. Other cell lines were in RPMI 1640 with 5% FBS plus penicillin/ streptomycin6cells/well (6-well dishes) were treated with vehicle or salinomycin at 48-hour post-seeding. Each point is average of three biological replicates. Annexin V-and propidium iodine(PI)-stained cells were analyzed by flow cytometry. Cells were stained using Annexin V Apoptosis Detection Kit APC . Samples were acquired on LSRII  within 30 minutes of conclusion of Annexin V staining. The Diva  or FlowJo  software was used for analysis.Viable cells (Trypan blue excluded) were counted in an automated cell counter . Cell number for each experiment was average from duplicate wells. Cells seeded at 10Cell Signaling Technology  was the source for antibodies to mTOR/ phospho-mTOR, Ser2448(D9C2); AKT1/phospho-AKT1, Thr308(#9275); Tuberin (TSC2)/phospho-Tuberin (TSC2), Thr1462(5B12); p70S6Kinase/phospho-p70S6 Kinase, Thr389 (#9205); GSK3α/β- phospho-GSK3β, Ser9 (#9322); S6 Ribosomal Protein/phospho-S6 Ribosomal Protein, Ser235/ 236(#2211); 4E-BP/phospho-4E-BP, Thr37/46(#9459); AMPKα/phospho-AMPKα, Thr172(40H9); Raptor/phospho-Raptor, Ser792 (#2083); PRAS40/phospho-PRAS40, Thr246(D4D2); ERK1/2/phospho-ERK1/2, Thr202/Tyr204 (D13.14.4E); Rheb (E1G1R); LC3B (D11). Antibodies to AR(N-20), FKBP12(N-19), GAPDH, β-actin and cofilin (FL-166: sc-33779) from Santa Cruz Biotech ; Antibody to phospho-AR, Ser-81(#07-1375) from EMD-Millipore . Antibodies to: CYP17A1 (EPR 6293) from GeneTex ; HSD3β1 (ARP 41821-P050) from Aviva Systems Biology  and p16  from Abcam .5′AAGA-CGCTTCTACCAGCTCACCAA-sens.e, 5′TCCC AGAAAGGATCTTGGGCACT-antisense; PSA: 5′TG ACCAAGTTCATGCTGTGT-sense, 5′GTCATTTCCAAGG TTCCAAG-antisense; NKX3.1: 5′AC-TTGGGGTCTTATC-sense, 5′CTTCTGCGGCTGCTTAGGG-antisense; CYP17A 1: 5′ACCTGGAGG TGCCAGATGAT-sense, 5′GGCG CACCTTGATCTTCACT-antisense; GAPDH internal control: 5′GA-CAGGATGCAGAAGGAGAT-sense, 5′TT GCTGATCCACATCTGCTG-antisense. A validated primer set, Bio-Rad (Cat#10025636) was used for qPCR of HSD3β1 cDNAs. QPCR was performed using CFX384 Real Time PCR machine .Total cell RNAs were isolated using TRIZOL® and converted to cDNAs for QRT-PCR analysis using previous conditions . PCR PriFor protein analysis, harvested cells were taken up in RIPA buffer  containing protease inhibitors . For phospho-protein analysis, protein phosphatase inhibitor  was added to RIPA buffer containing protease inhibitors. Cells were sonicated  using Sonic Dismembrator, setting 5 . Supernatants of lysed cells were analyzed by Western blotting after 10% SDS-PAGE . Two seg6 cells, mixed 1:1 (v/v) with matrigel . Treatment started at ~200-250 mm3 of palpable tumor. Tumor volume=½(length × width2); length=maximum longitudinal diameter and width=maximum transverse diameter, measured with a caliper. Tumors with an adjacently placed ruler were photographed for record keeping. Mice received salinomycin  and vehicle  by i.p. injections (LNCaP-II xenografts) or oral gavage (C4-2 xenografts). Tumor lysates were prepared in RIPA buffer. Protocol approved by the Institutional Animal Care and Use Committee was used.Tumor xenografts were produced subcutaneously at the right flank of athymic nude mice  with cells at 3 × 10Data were analyzed using SPSS 15 software. Student's t-test was used; data presented as mean ± SEM; p < 0.05 is significant.


Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPAs), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor PDCD4 and MSH2 genes, by down regulating micro RNA-21 (miR-21). In GBMs miR-21 expression is significantly elevated and is associated with chemo and radioresistance. Both MPS1 and miR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of miR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind miR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-β/SMAD signaling via miR-21 regulation. We also, show the prognostic effect of miR-21, PDCD4 and MSH2 levels to patient survival across different GBM molecular subtypes. This scenario in which miR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy. Glioblastoma multiforme or glioblastoma (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Patients have a median survival of less than 15 months following standard of care , 3. PrevBeyond mitosis MPS1 kinase has been implicated, in genotoxic stress response, such as stress caused by DNA damage , 11, in In the present study using Reverse phase protein arrays (RPPAs) and bioinformatics approach, we assessed the MPS1 mediated cell signaling pathways in GBM. Our results demonstrate that MPS1 inhibition results in induction of tumor suppressor PDCD4 and MSH2 expression through modulation of oncogenic miR-21 via a non-canonical Smad signaling pathway.P < 0.05) differentially expressed proteins between normal and MPS1 inhibited cells   and MSH2  levels after miR-21 knockdown  repressed induction of PDCD4 and MSH2    repressed IR induced miR-21 levels in U251 GBM cells  decrease in GBM cell proliferation (both U251 and U87) with MPS1  but not totally rescued GBM cell attenuation. Notably, ectopic expression of miR-21 mimic alone had no negative effect on GBM cell growth  were randomized into two groups, vehicle treated controls and NMSP715 100 mg/kg . One day after drug treatment the mice were sacked and tumors were isolated at different time points. We noticed significant (P < 0.05) increase in PDCD4 and MSH2 levels at later time points with NMSP715 treatment  decrease in phospho (p) SMAD3 (but not pSMAD2) both under RNAi and NMSP715 mediated inhibition of MPS1. Immunofluorescence studies also show an increase in PDCD4 levels and decrease in pSMAD2/3 levels in the nucleus of MPS1 inhibited U251 cells . Using in house Glioblastoma Bio Discovery Portal (GBM-BioDP-(http://gbm-biodp.nci.nih.gov)) [n = 197), which clearly show miR-21 anti-correlated to PDCD4, MSH2 expression ) , multivapression . These rp < 0.05) rescued anti proliferative effect of MPS1 inhibition on GBM cells  decrease in phospho (p) SMAD3 levels (but not pSMAD2) with MPS1 inhibition in GBM cells  human GBM cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM)  with 10% fetal bovine serum (FBS), and maintained at 37°C, 5% CO2. NMSP715 was obtained from Calbiochem., U.S.A (cat: 475949). Drug was reconstituted in dimethyl sulfoxide (DMSO) and stored at -20 C.As described earlier (5) siRNA transfections, 2-pmol siMPS1, )  was complexed with RNAi Max lipid transfection reagent (Invitrogen) in DMEM media for 15 minutes at ambient temperature. Two thousand cells suspended in DMEM supplemented with 20% FBS were then added. . Plates were maintained at ambient temperature for 15 minutes before being placed at 37 C/5% CO2. Cell viability was assessed five days post siRNA transfection through quantification of ATP . Untransfected cells and wells transfected with negative  and positive  control siRNAs were used as controls. Proteins for Western blot analysis was harvested 48 hours post siRNA transfection or Drug treatments.U251, U87 GBM cells were transfected as above with 3 μl Lipofectamine RNAiMAX  mixed with 30 nM meridian mimic hsa-miR-21-5p (cat: C-301023-01-0002: UAGCUUAUCAGACUGAUGUUGA) or negative control mimic (cat: CN-002000-01-05) . hsa-miR-21-5p targets PDCD4 and MSH2 (TarBase) .http://www.ncbi.nlm.nih.gov/geo/ (GSE67502). Further Ingenuity Pathway Analysis (IPA) software (http://www.ingenuity.com) was used to analyze cellular signaling pathways for each group of genes under MPS1 inhibition, to predict Top Canonical Pathways and their associated regulators.Treated GBM cell  lysates were prepared in RPPA lysis buffer [1% Triton X-100, 50 nmol/L Hepes (pH 7.4), 150 nmol/L NaCl, 1.5 nmol/L MgCl2, 1 mmol/L EGTA, 100 nmol/L NaF, 10 nmol/L NaPPi, 10% glycerol, 1 nmol/L phenylmethylsulfonyl fluoride, 1 nmol/L Na3VO4, and aprotinin 10 μg/mL) as described elsewhere (37), and sent to RPPA Core Facility, MD Andersen Cancer Center, Houston, TX for RPPA analysis. Briefly, 5 serial dilutions of lysates were arrayed on nitrocellulose-coated slides, probed with (172 phosphorylated and non- phosphorylated) antibodies, and visualized by DAB colorimetric reaction . Relativhttps://tcga-data.nci.nih.gov). An in-house Glioblastoma Bio Discovery Portal (GBM-BioDP) [Verhaak et al [p-value. A log-rank of < = 0.05 and a p-value < = 0.05 were considered statistically significant. This tool also helped us to construct expression correlation heat maps between miRNAs and gene products. GBM-BioDP is a free web-accessible resource that hosts a subset of the glioblastoma TCGA data and enables an intuitive query and interactive display of the resultant data (http://gbm-biodp.nci.nih.gov) [Prognostic effect of miR-21, PDCD4, MSH2 expression correlated to patient survival for an independent cohort of 197 GBM patients was analyzed by curating GBM-TCGA database (M-BioDP)  was usedak et al ) and potnih.gov) .Total RNA from GBM cells was extracted using Trizol (Invitrogen), and cDNA was synthesized by using miScript II RT kit  as per manufacturer recommendations. Expression of miR-21-microRNA (Hs04231424_s1) was determined by the TaqMan miRNA-assay , and normalized using the 2^^CT-method relative to U6-snRNA (Hs00984809_m1). MPS1-mRNA was quantified by TaqMan-qRT–PCR and normalized to GAPDH (AppliedBiosystems) . All TaqMan-PCRs were performed in triplicates run on Applied Biosystems 7500 Real Time PCR thermal cycler.Cell pellets were lysed on ice in RIPA buffer  supplemented with Complete Mini EDTA-free Protease Inhibitor Cocktail  and Phosphatase Inhibitor Cocktail . (Tumors were lysed in the same buffer using a Homogenizer). Protein concentrations were determined by Bradford assay . Protein(50ug) was diluted 1:5 in 5X protein loading buffer , boiled at 80°C for 5 minutes, electrophoresed on a 4-20% Tris-Glycine gel, and transferred using a Trans-Blot Turbo Transfer System . Membranes were blocked in 5% Non-fat milk powder (BioRad), incubated with primary antibody overnight at 4°C, incubated with HRP-coupled secondary antibody 1 hour at room temperature, developed with Visualizer Western Blot Detection Kit , and visualized on a LAS-4000 imager . The following antibodies were used at 1:1000 dilutions: rabbit anti-Smad2/3 (#3102), rabbit-anti- Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828), mouse-anti- MSH2 (#2850), rabbit-anti-PDCD4 (#9535) were from Cell Signaling Technology (CST)., MA, human-anti-MPS1 ; mouse anti-actin . Secondary antibodies, goat anti-rabbit-HRP and goat anti-mouse-HRP  were used at 1:10,000 dilution. Protein band Intensities were quantitated using Image-J software (NIH).Localization of endogenous pSMAD2/3 and PDCD4 was assessed by immunofluorescent staining. U251 GBM cells were seeded on 4 well chamber slides  . After attachment and treatment as indicated, cells were fixed in 4% paraformaldehyde (in PBS +0.1% Triton X-100 for permeabilisation)  and then blocked with 3% BSA in PBS, followed by overnight incubation with goat polyclonal-anti- pSMAD2/3 ) and rabbit-anti-PDCD4 primary antibody  (1:500 dilution with PBS containing 3% BSA). Alexa flour conjugated AffiniPure donkey anti-goat IgG and goat-anti-rabbit IgG secondary antibodies (Invitrogen) were then applied. Slides were then mounted with fluorescent medium VECTASHIELD containing DAPI (Nuclear stain)  to preserve the fluorescent signal. The localization of endogenous pSMAD2/3 and PDCD4 were visualized and images captured with Olympus FSX100 fluorescent microscope.P* < 0.05. Analysis was done using MS Excel 2010 software .Data presented are the mean ± S.D from three independent experiments unless indicated otherwise. All statistical tests were two-sided. For comparisons between groups, a Student's t test was performed and the level of significance was set at


While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT.Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to  Hematopoietic stem cells (HSC) originate in the Aorta-Gonad-Mesonephros (AGM) region of the embryo –4. A subtrans with Notch receptor 1 to 4 on an adjacent cell. Upon this interaction, the intracellular domain of the receptor is cleaved and translocated to the nucleus where it activates the CSL complex, resulting in the induction of Notch target genes [cis-inhibition [We have shown that HoxA3, a transcription factor of the homeodomain family , 10, bloet genes . Activathibition –16.in vivo [Activation of the Notch pathway is necessary for proper vasculogenesis and angiogenesis in vivo –19 and fin vivo –23. In ain vivo . This isin vivo . It has in vivo . While tcis-inhibition and blocked the initiation of blood commitment. Consistent with our model, forced activation of Notch pathway, either in a ligand-independent fashion or by down-regulation of Jag1, triggered blood commitment by down-regulating the endothelial cell phenotype, a step required for the initiation of EHT. These findings suggest that HoxA3 prevents blood commitment through Notch cis-inhibition and identify the Notch pathway as a potential therapeutic target for blood development in vitro.Here, we identify a new mechanism by which HoxA3 tightly controls blood formation by inhibiting the Notch pathway in HE. We show that HoxA3 induced up-regulation of Jag1 in HE that has not yet started blood commitment. This upregulation caused Notch ligand 2. Embryoid Bodies (EBs) were differentiated by hanging-drops suspension culture in EB differentiating media . At day 4 of differentiation, EBs were treated with doxycycline (1μM) to induce HoxA3 overexpression. At day 6, EBs were collected, dissociated by using trypsin at 37°C for 2–3 min following mechanical dissociation, and immunostained with Flk1-PE and Ve-cadherin-APC antibodies (2. Notch inhibition was performed with DAPT treatment (20 and 60 μM). For experiments involving Notch ligand overexpression, OP9-Dll1 [HoxA3-inducible murine ES cells were previously generated, by cassette exchange recombination in doxycycline-inducible locus . ES celltibodies . Two hunOP9-Dll1  and OP9 The pMSCV-hNICD-Ires-GFP was constructed by subcloning a blunted Not1/Ecor1 hNICD (from NOTCH1 receptor) fragment  into pMSRetroviral transduction of pMSCV-ires-GFP, pMSCV-hNICD-ires-GFP and pLKO-Jag1 KD were performed as reported . RetroviTotal RNA was extracted with Trizol LS (Invitrogen) and cDNA generated with iScript reagent (BioRad) according to protocol. PCR was performed using TaqMan mix (Life Technologies) on a 7600 RT-PCR System from Applied Biosystems. Probes purchased from Applied Biosystems are listed in Flow cytometry was performed by staining with specific anti-mouse antibodies. Analysis was performed using FACS ARIA III and data analyzed using FlowJo software. For Immunofluorescence staining, cells were fixed in 4% PFA for 4 min following by antigen retrieval in Tris-EDTA buffer pH 9.0 and permeabilized as reported in 0.2% triton for 15 min . Cells w293T cells were transfected with pMSCV-hNICD-iresGFP or pMSCV-iresGFP, proteins were extracted with RIPA Buffer (Millipore # 20–188). After blotting membranes were stained with Ponceau S and probed with anti-Myc-tag for NICD detection. Cytoplasmic proteins from Bend3 cells transfected with pLKO Jag1-KD or pGIPZ-GFP and Day 6 FV progenitors were extracted using NE-PER extraction kit (Thermo-Scientific #78833). After protein separation and blotting (Millipore #456–1096 and # 170–4158) membranes were probed using Jag1 specific antibody followed by specific secondary antibody. Membranes were imaged using BioRad Chemi Doc Imaging system. All antibodies used are listed in Data were analyzed using GraphPad Prism software . Data obtained from 6 hours, 5 days of HoxA3 up-regulation experiments and DAPT treatment on the expression of Notch target genes in BEND3 cells were analyzed with unpaired, two-tailed t-test. Independent data sets were analyzed by two-way ANOVA using HoxA3 regulation (Con vs HoxA3) and treatment  as between-subject followed by Fisher’s LSD post hoc tests. One-way ANOVA was performed to analyze the effect of Jag KD on Con and HoxA3 upregulation. Significance was set at p<0.05.We have previously reported an experimental model that recapitulates blood formation from EHT using embryonic stem cell differentiation. This model takes advantage of the ability of HoxA3 to block blood formation from HE, providing a synchronized and homogeneous population of HE  S1A FigS1A Fig. in vitro using the OP9 co-culture. The starting population of endothelial cells expressed CD31 and Tie2 (not shown) as well as Flk1 and Ve-cadherin  but not in cis .To test whether the increased expression of Jag1 in endothelial cells had a biological effect in g HoxA3, . Togethe+/Cxcr4+ and Ve-Cadherin+/CD44+)  . More imal cells . These dNext, we tested whether the blockade of blood formation was due to HoxA3-dependent repression of Notch activation. To do this, we forced activation of Notch in a ligand-independent fashion using NICD transduction . NICD up+/Ve-Cadherin+ endothelial cells , while unable to induce blood formation (not showed), induced an increase of Notch target genes even if HoxA3 was overexpressed confirming that Jag1 up-regulation in HE was cis-inhibiting the Notch pathway  Representative flow cytometric profile of endothelial surface markers Flk-1/Ve-Cadherin and hematopoietic surface markers c-Kit/CD41, of 10000 cells obtained from dissociated day 6 EBs treated with or without Dox at day 4 of differentiation. B) Representative flow cytometric profile and frequency quantification of arterial surface markers Ve-Cadherin/CD44 and Ve-Cadherin/CXCR4 on 200,000 EB-derived Flk1+/VE-cadherin+ cells without or with HoxA3 overexpression and co-cultured on OP9 for 5 days. C) Gene expression levels in purified endothelial cells co-cultured with OP9 for 5 day without or with doxycycline-induced HoxA3 overexpression. Arterial, EfnB2 and vein markers EphB4 are plotted. D) Representative flow cytometric profile and frequency quantification of hematopoietic surface markers c-Kit/CD41, and c-Kit/CD45 on 200,000 EB-derived Flk1+/VE-cadherin+ cells without or with HoxA3 overexpression and co-cultured on OP9 for 5 days E) Assessment of Notch pathway activation on OP9 cells alone (left) or purified OP9 cells after co-culture with Flk1+/VE-cadherin+ without or with HoxA3 overexpression (right). Notch target genes Hes1 and Hey2 are plotted. Where present asterisks (*) identify significant paired two-tailed T test (* p<0.05). Statistical analysis is reported on S2 Table.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigA) Quantification of frequencies of hematopoietic surface markers  on 200,000 EB-derived Flk1+/VE-cadherin+ cells without or with HoxA3 overexpression and co-cultured on OP9 for 5 days in the presence or absence of the Notch inhibitor DAPT (20μM) B) Evaluation of Notch pathway inhibition  on endothelial cells (BEND3) treated with 20μM of DAPT or DMSO (CON). C) Frequency quantification of 200,000 cells Flk1+/VE-cadherin+ obtained from day 6 EBs and co-cultured on OP9 for 5 days with or without HoxA3 overexpression and treated without (DMSO/CON) or with 20μM of DAPT. Hematopoietic surface markers Gr1-CD45 and arterial/vein Ve-Cadherin, CXCR4 and CD44 and are plotted. Statistical analysis is reported on S3 Table.(PDF)Click here for additional data file.S4 FigA) Western blot analysis and Ponceau S staining of the indicated proteins (cMyc-NICD and GAPDH) and total loading protein, respectively, in 293T cells transfected with pMSCV-hNICD-ires GFP plasmid (NICD-1/NICD-2), backbone vector pMSCV-ires GFP (CON) and non-viral infection (NVI). B) Frequency quantification of endothelial markers VeCadherin and Pecam (CD31), from gated GFP positive cells transduced with pMSCV-iresGFP (CON) or with pMSCV-hNICD1-IresGFP (NICD) and co-cultured on OP9 for 5 days in absence (CON) or presence (HoxA3) of HoxA3 overexpression. C) Quantification of frequencies of hematopoietic surface markers ckit, CD41, CD45, and D) representative flow cytometric profile of myeloid markers CD45, Gr1 and Ter119 on 200,000 cells Flk1+/VE-cadherin+ obtained from day 6 EBs, transduced with pMSCV-iresGFP (CON) or with pMSCV-hNICD1-IresGFP (NICD) and co-cultured on OP9 for 5 days in absence (CON) or presence (HoxA3) of HoxA3 overexpression. E) Frequency quantification and representative flow cytometric profile, of 200,000 cells Flk1+/VE-cadherin+ obtained from day 6 EBs, transduced with pMSCV-iresGFP (CON) or with pMSCV-hNICD1-IresGFP (NICD) and co-cultured on OP9 for 5 days in absence (CON) or presence (HoxA3) of HoxA3 overexpression. Viability markers PI and Annexin V are plotted. Post-hoc analysis are reported as asterisks (*) alone represents significant differences compared to CON/Dox-, * p<0.05, and bars represents significant differences (*) between indicated groups, p<0.05. Statistical analysis is reported on S4 Table.(PDF)Click here for additional data file.S5 FigA) Quantification of frequencies of endothelial surface markers Flk-1+/Ve-Cadherin+ obtained from 200,000 EB-derived Flk1+/VE-cadherin+ cells and co-cultured on OP9 control (CON) or OP9 overexpressing Dll1 (OP9-Dll1) for 5 days in Control or HoxA3-overexpressing HE cells. B) Quantification of frequencies of hematopoietic surface markers  on cells obtained from day 6 EBs, transduced with empty vector (CON) or with shRNA-Jag1-GFP (JKD) and co-cultured on OP9 for 5 days in Control (Con) or HoxA3 overexpression.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableA) Two tails T-test analysis of Notch components on control endothelial cells (CON) compare to endothelial cells derived from 6 hours upregulation of HoxA3 in D6 total EBs (HoxA3) B) Two tails T-test analysis of Notch components endothelial derived cells (EDC) co-cultured with OP9 for 5 days without (CON) or with HoxA3 overexpression.(PDF)Click here for additional data file.S3 Table2-way ANOVA analysis of endothelial derived cells co-cultured with OP9 for 5 days without (CON) or with HoxA3 overexpression and treated without (DMSO) or with (DAPT) Notch inhibitor.(PDF)Click here for additional data file.S4 Table2-way ANOVA analysis of endothelial derived cells transduced with pMSCV-NICD-ires GFP (NICD) or pMSCV-iresGFP (CON) and co-cultured with OP9 for 5 days without (CON) or with HoxA3 overexpression.(PDF)Click here for additional data file.S5 Table2-way ANOVA analysis on endothelial derived cells co-culture with OP9-CON vs OP9-Dll1 for 5 days without (CON) or with HoxA3 overexpression.(PDF)Click here for additional data file.S6 Table2-way ANOVA analysis of endothelial derived cells transduced with pGIPZ (CON) or plKO.1-Jag1 KD-ires GFP (JAG KD) and co-cultured with OP9 for 5 days without (CON) or with HoxA3 overexpression.(PDF)Click here for additional data file.


Dear Editor,CRISPR-Cas9  systems have been harnessed for kinds of genome manipulation, including gene editing, transcription regulation, and chromosome loci imaging  assay using a Cy5-tagged telomere-specific-probe and immunofluorescence staining assay using a primary antibody against HA tag (each fluorescence protein contains a HA tag) and an Alexa488-labeled secondary antibody were performed in the same cells , while there was no statistical significant difference between sfGFP and mNeonGreen , and GCN4 peptide binding single-chain variable fragment antibody (scFv-GCN4) was fused to superfolder-GFP (sfGFP), mNeonGreen, or three-tandem-repeats of mNeonGreen (3XmNeonGreen). As sgRNA structure could significantly affect imaging quality, a sgRNA scaffold optimized by A-U base pair flip and hairpin extension was used to eliminate protein aggregation and increase bright dot number Supplementary material 2 (AVI 471 kb)Supplementary material 3 (AVI 661 kb)Supplementary material 4 (AVI 94 kb)Supplementary material 5 (AVI 293 kb)Supplementary material 6 (AVI 97 kb)Supplementary material 7 (PDF 270 kb)Below is the link to the electronic supplementary material.


LMNA) gene, resulting in the production of a truncated protein product—progerin. WS is caused by mutations in WRN gene, encoding a loss-of-function RecQ DNA helicase. Here, by gene editing we created isogenic human embryonic stem cells (ESCs) with heterozygous (G608G/+) or homozygous (G608G/G608G) LMNA mutation and biallelic WRN knockout, for modeling HGPS and WS pathogenesis, respectively. While ESCs and endothelial cells (ECs) did not present any features of premature senescence, HGPS- and WS-mesenchymal stem cells (MSCs) showed aging-associated phenotypes with different kinetics. WS-MSCs had early-onset mild premature aging phenotypes while HGPS-MSCs exhibited late-onset acute premature aging characterisitcs. Taken together, our study compares and contrasts the distinct pathologies underpinning the two premature aging disorders, and provides reliable stem-cell based models to identify new therapeutic strategies for pathological and physiological aging.Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) are two of the best characterized human progeroid syndromes. HGPS is caused by a point mutation in lamin A ( LMNA mutation (p.G608G/+). LMNA encodes A-type lamins that belongs to the family of nuclear lamina proteins, and a point mutation (p.G608G) in LMNA creates an aberrant splicing site in exon 11, resulting in the production of a truncated protein, progerin  and Werner syndrome (WS) are best characterized , homozygous (LMNAG608G/G608G) LMNA mutation, and homozygous WRN deficiency (WRN−/−) under the same genetic background , we generated heterozygous and homozygous nes Fig. A. Combinund Fig. B–D. All und Fig. B and 2A.ion Fig. B and 2B.ion Fig. D. In addClinical observations in HGPS and WS patients indicate that premature aging disorders are often accompanied with defects in mesenchymal lineages, such as lipodystrophy, osteoporosis and atherosclerosis  and homozygous (LMNAG608G/G608G) HGPS-MSCs grew at normal rate up to passage 6. Differences in cell cycle distribution were also observed between HGPS-MSCs and WS-MSCs , HGPS-ESCs and WS-ESCs were differentiated into HGPS-ECs and WS-ECs, respectively. CD31 and CD144 double-positive cells were sorted , these cells may be particularly amenable to mechanistic studies using multi-omics techniques.In addition, we observed that the homozygous HGPS-MSCs exhibited more severe aging phenotypes with a higher level of progerin than the heterozygous MSCs. Thus, the MSCs with homozygous or heterozygous LMNA mutation or WRN deficiency are cell-type-specific  and three genetically edited ESCs were maintained on mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder in human ESC culture medium: 80% DMEM/F12 (Gibco), 20% Knockout Serum Replacement (Gibco), 0.1 mmol/L non-essential amino acids , 2 mmol/L GlutaMAX (Gibco), 55 μmol/L β-mercaptoethanol (Invitrogen), and 10 ng/mL FGF2 ; ESCs were also cultured on Matrigel (BD Biosciences) with mTeSR medium (STEMCELL Techonologies). All MSCs were cultured in MSC culture medium: 90% α-MEM + Glutmax (Gibco), 10% fetal bovine serum , 1% penicillin/streptomycin (Gibco) and 1 ng/mL FGF2 . ECs were cultured in EGM2 medium (Lonza).LMNA G608G knock-in was generated same as previous report with some modifications   for Briefly, WT-ESCs and three genetically edited ESCs were plated on Matrigel in EC differentiation medium I  for 3 days. Differentiation medium II  was used for another 3 days then CD31/CD144 double-positive cells were sorted by FACS.MSCs were differentiated as previously described  as previously described. In brief, PCR products were purified by using gel extraction kit (Qiagen), and subsequently cloned into the pMD20 T vector (Transgen). 7 clones from each sample were sequenced with the universal primer M13.Bisulfite treatment of DNA was carried out by using EZ DNA Methylation Kit (Zymo Research) following the manufacturer’s instructions. About 1 μg of genomic DNA was used. A genomic fragment of the Primers used for PCR: meF-OCT4, 5′-ATTTGTTTTTTGGGTAGTTAAAGGT-3′; meR-OCT4, 5′-CCAACTATCTTCATCTTAATAACATCC-3′.6 ESCs were administrated subcutaneously into NOD/SCID mice . 6–12 weeks after injection, mice were killed and teratomas were analyzed by immunostaining. All animal experiments were conducted with approval of the Institute of Biophysics, Chinese Academy of Science.Teratoma assay was performed as described  and washed by PBS twice. Cells were incubated with primary antibody diluted with 10% FBS in PBS for 1 h at room temperature and then sorted by using a flow cytometer (BD FACSAria IIIu).Antibodies used for cell sorting: anti-CD73 (550741), anti-CD90 (555595), anti-CD31 (555445), anti-CD144 (560410) antibodies were from BD Biosciences; anti-CD105 (17–1057) antibody was from eBioscience.6 cells were processed with the Click-iT EdU Flow Cytometry Assay Kits (Invitrogen) according to the manufacturer’s instructions. In brief, the cells were harvested after 2 h incubation with EdU and stained with Alexa Fluor 647 dye azide and propidium iodide. Cells were examined by fluorescence-activated cell sorting (FACS) using a flow cytometer (BD LSRFortesa).For cell cycle analysis, about 1 × 10The single-cell clonal expansion assay was carried out as described , and then apoptotic cells were quantified by FACS. For NO detection, cells were treated with DAF-FM (Molecular Probes) for 30 min and quantified by FACS.In brief, ECs were collected after 6 h incubation with Dil-Ac-LDL (Molecular Probes) in EC culture medium. For FACS analysis, cells were collected by using TrypLE Express (Gibco) and measured by a flow cytometer (BD LSRFortesa). For immunofluorescence detection, cells were processed following the immunofluorescence microscopy protocol.4 cells were suspended in 500 μL EC medium and then seeded on Matrigel coated plate. After 6–8 h, lattice-like vessel structures formed and then cells were stained with Calcein-AM (Invitrogen) and examined by using fluorescence microscope (Olympus).Briefly, 5 × 106 cells were lysed in 100 μL RIPA buffer  supplemented with NaF, NaVO4 and a protease-inhibitor mixture (Roche). Typically 20 μg of proteins were separated by SDS-PAGE, transferred to a PVDF membrane (Millipore), and blotted with one of the following primary antibodies and then HRP-conjugated secondary antibodies. The quantification of western blot was performed with Image Lab software for ChemiDoc XRS system (Bio-Rad).1 × 10Primary antibodies for western blotting include anti-WRN , anti-Progerin , anti-P21 , anti-LAP2β , anti-HP1α , anti-Actin , anti-Lamin B1 , anti-P16 , anti-H3K9me3 , anti-β-Tubulin .Total RNA was extracted by using TRIzol reagent (Invitrogen). 2 μg of RNA was converted to cDNA by using GoScript Reverse Transcription System (Promega), and 1/50 volume of the cDNA reaction was applied to PCR using primers for human GAPDH, Progerin. RT-qPCR was performed by using iTaq Universal SYBR Green Supermix (Bio-Rad).Primers used for RT-PCR: GAPDH-F, 5′-TCGGAGTCAACGGATTTGGT-3′; GAPDH-R, 5′-TTGCCATGGGTGGAATCATA-3′; Progerin-F, 5′-ACTGCAGCAGCTCGGGG-3′; Progerin-R, 5′-TCTGGGGGCTCTGGGC-3′.Cells seeded on microscope coverslips were fixed with 4% formaldehyde in PBS for 30 min, permeabilized with 0.4% Triton X-100 in PBS for 25 min, and blocked with 10% donkey serum in PBS for 1 h. The coverslips were incubated with primary antibody (diluted with 1% donkey serum in PBS) overnight at 4°C and then incubated with fluorescence-labeled secondary antibody (diluted with 1% donkey serum in PBS at 1:500) at room temperature for 1 h. Hoechst 33342 (Invitrogen) was used to stain nuclear DNA.Antibodies for immunofluorescence were purchased from the following companies. Abcam: anti-NANOG (ab21624), anti-Nucleolin (ab22758); ZSGB-Bio: anti-hSMA (ZM-0003); Sigma: anti-TUJ1 (T2200); Santa Cruz Biotechnology: anti-OCT4 (sc-5279), anti-SOX2 (sc-17320), anti-Progerin (sc-81611), anti-Lamin B (sc-6217); Cell Signaling Technology: anti-HP1α (2616), anti-FOXA2 (8186S), anti-CD144 (2158); Bethyl Laboratories: anti-53BP1 (A300-273A); Millipore: anti-γ-H2AX (05-636); BD Bioscience: anti-LAP2β (611000), anti-eNOS (610296); Vector: anti-Ki67 (VP-RM04); Dako: anti-vWF (A082).t-test was used to analyse differences between different cell lines. Results were presented as mean ± SEM. P values < 0.05, P values < 0.01 and P values < 0.001 were considered statistically significant .Student’s


NPC-26 is novel mitochondrion-interfering compound. The current study tested its potential effect against colorectal cancer (CRC) cells. We demonstrated that NPC-26 induced potent anti-proliferative and cytotoxic activities against CRC cell lines . Activation of AMP-activated protein kinase (AMPK) signaling mediated NPC-26-induced CRC cell death. AMPKα1 shRNA knockdown or dominant negative mutation abolished NPC-26-induced AMPK activation and subsequent CRC cell death. NPC-26 disrupted mitochondrial function, causing mitochondrial permeability transition pore (mPTP) opening and reactive oxygen species (ROS) production. ROS scavengers (NAC or MnTBAP) and mPTP blockers (cyclosporin A or sanglifehrin A) blocked NPC-26-induced AMPK activation and attenuated CRC cell death. Significantly, intraperitoneal injection of NPC-26 potently inhibited HCT-116 tumor growth in severe combined immuno-deficient (SCID) mice. Yet, its anti-tumor activity was significantly weakened against AMPKα1-silenced HCT-116 tumors. Together, we conclude that NPC-26 kills CRC cells possibly via activating AMPK signaling. Colorectal cancer (CRC) is still a major malignancy in the world, which causes significant mortality each year –3. Mitocet al., demonstrated that itraconazole directly binds to and inhibits mitochondrial protein voltage-dependent anion channel 1 (VDAC1), causing ATP depletion and AMP:ATP ratio increase   were utiThe above shRNA results imply that AMPK activation mediates NPC-26-induced cytotoxicity against HCT-116 cells. To further support this hypothesis, a dominant negative AMPKα1  construct , 31, 32 et al., [A very recent study by Dong et al.,  demonstret al., )  or mPTPet al.,  and sanget al., . Remarkain vivo, the severe combined immuno-deficient (SCID) mice bearing HCT-116 xenograft tumor model was applied. Results in Figure i.p. injection of NPC-26  [in vivo was remarkably weakened against HCT-116 tumors with AMPKα1 shRNA (“Seq-1”)  induced significant AMPK activation, or p-AMPK/p-ACC, in control tumors   dramatic) Figure , indicats Figure . NotablyAMPK plays a pivotal role in regulating a number of key cellular functions, from energy metabolism, cell mitosis, apoptosis to autophagy , 38. Howetc.). In fact, the activity of AMPK could increase over 100-fold on phosphorylation of a conserved threonine residue (Thr-172) within the activation loop at α1 subunit [Under certain circumstances, it has yet been proposed that AMPK activation could also be pro-survival , 42. The subunit , 43. In It should be noted that mitochondria in cancer cells are structurally and functionally different from those in normal  cells, which are often highly-active in malignant cells to participate in metabolic reprogramming and cell activities , 5. IntrIntriguingly, AMPK blockage, via AMPKα1 shRNA knockdown or Thr-172 dominant negative mutation, didn't completely abolished NPC-26-mediated killing of CRC cells Figure . MeanwhiNPC-26 was a gift from Dr. Dong's group . N-acetyHCT-116, DLD-1 and HT-29 CRC cell lines were provided by Dr. Lu's group , 46. CelTo test cell survival, cell counting kit-8  assay kit was performed based on the attached manual. CCK-8 absorbance optic density (OD) was recorded at 450 nm.After treatment, trypan blue (0.2%)-stained cells was counted by the Countess automatic cell counter . The cell death percentage (%) was calculated by the number of the trypan blue cells divided by the total cell number.To test cell proliferation, 5-bromo-2’-deoxyuridine (BrdU) incorporation assay was applied. Briefly, BrdU  was pre-added. Following treatment of cells, BrdU incorporation was determined in enzyme-linked immunosorbent assay (ELISA) format. ELISA OD at 450 nm was tested.HCT-116 cells with applied NPC-26 treatment were re-suspended in DMEM medium containing 0.5% agar (Sigma), which were then plated onto a pre-solidified six-well plate. Afterwards, cells were cultured in NPC-26-containing medium for 8 days. The remaining colonies were counted manually.Cell or HCT-116 tumor tissue samples were incubated in lysis buffer described , 46. QuaAs described previously , mitochoFollowing treatment, cells were stained with CellRox Orange Reagent  at 37°C for 30 min. ROS content was detected by the above fluorescence microplate reader .Three AMPKα1 shRNAs  with nonDominant-negative (dn) mutant of AMPKα1 (AMPKα1-T172A) construct was provided by Dr. Lu's group , 31, 32.6 cells per mouse), expressing scramble control shRNA or AMPKα1 shRNA (“Seq1”), were subcutaneously (s.c.) injected into SCID mice. Within three weeks, the xenografted tumors were established around 100 mm3 in volume. The SCID mice were then treated as described. Tumor volumes, recorded every 5 days, were calculated using the formula: (mm3) = (A2 × B)/2: A and B were the shortest and the longest diameter, respectively. Mice body weights were also recorded.The protocols using 6-8 week-old severe combined immunodeficient (SCID) mice (weighting 18-19g) were approved by the IACUC of all authors’ institutions. Exponentially growing HCT116 cells (5*10The staining was performed on the cryostat sections (4 μm) of HCT-116 tumors using the standard protocol . p-AMPKαp-value, calculated by ANOVA, of less than 0.05 was considered statistically significant.All values were expressed as the mean ± standard deviation (SD). A


Scientific Reports 10.1038/s41598-017-04720-7, published online 10 July 2017Correction to: This Article contains typographical errors in the Acknowledgements section.“We acknowledge support from the Austrian Science Fund (FWF) project P23846-N16,”should read:“We acknowledge support from the Austrian Science Fund (FWF) project P26253-N27”


C. elegans, death-associated protein kinase (DAPK-1) regulates epidermal morphogenesis, innate immunity and wound repair. Combining genetic suppressor screens and pharmacological tests, we find that DAPK-1 maintains epidermal tissue integrity through regulation of the microtubule (MT) cytoskeleton. dapk-1 epidermal phenotypes are suppressed by treatment with microtubule-destabilizing drugs and mimicked or enhanced by microtubule-stabilizing drugs. Loss of function in ptrn-1, the C. elegans member of the Patronin/Nezha/CAMSAP family of MT minus-end binding proteins, suppresses dapk-1 epidermal and innate immunity phenotypes. Over-expression of the MT-binding CKK domain of PTRN-1 triggers epidermal and immunity defects resembling those of dapk-1 mutants, and PTRN-1 localization is regulated by DAPK-1. DAPK-1 and PTRN-1 physically interact in co-immunoprecipitation experiments, and DAPK-1 itself undergoes MT-dependent transport. Our results uncover an unexpected interdependence of DAPK-1 and the microtubule cytoskeleton in maintenance of epidermal integrity.Epidermal barrier epithelia form a first line of defense against the environment, protecting animals against infection and repairing physical damage. In DOI:http://dx.doi.org/10.7554/eLife.15833.001 Death-associated protein kinase 1 (DAPK1) and its related calcium-regulated serine/threonine kinases play a wide variety of roles in cell death and tumor suppression . MammaliC. elegans the sole DAPK family member, DAPK-1, plays roles in autophagy and in excitotoxic neuronal death  regulators in the mature epidermis. Genetic and pharmacological manipulations suggest aberrant dapk-1 function causes excessive MT stabilization, resulting in morphological defects. dapk-1 epidermal defects can be suppressed by loss of function in the MT minus end binding protein PTRN-1 and by pharmacological destabilization of MTs. Moreover, overexpression of the MT-binding domain of PTRN-1 is sufficient to induce dapk-1-like epidermal defects. Our data suggest DAPK-1 destabilizes epidermal MTs by inhibiting the function of PTRN-1. We further show for the first time that DAPK-1 itself undergoes MT-dependent transport. Our findings reveal an unexpected interplay between DAPK-1, the epithelial MT cytoskeleton, and epidermal morphology and wound repair.Here we took a genetic approach to understanding DAPK-1’s functions in the epidermis. We identified genetic suppressors and enhancers of dapk-1 interactors, we screened for genetic suppressors of dapk-1 epidermal phenotypes. All dapk-1 mutants display epidermal morphological (Mor) defects, with penetrance varying depending on the allele (dapk-1(ju4), which causes a missense alteration S179L in the DAPK-1 kinase domain, causes 100% of animals to display this aberrant morphology. We mutagenized dapk-1(ju4) animals and screened for suppression of the Mor phenotype (see Methods). We identified multiple extragenic suppressors of dapk-1(ju4), two of which are described here. One suppressor, ju698, causes a nonsense mutation in ptrn-1, which encodes the C. elegans member of the Patronin/CAMSAP/Nezha family of MT minus end binding proteins. A null allele ptrn-1(lt1) suppressed dapk-1(ju4) phenotypes to the same extent as ptrn-1(ju698) (dapk-1(ju4) by ptrn-1(0) was rescued by a single-copy insertion (Mos-SCI) ptrn-1(+) transgene and by transgenes expressing PTRN-1 under the control of the dpy-7 promoter, specific to the larval epidermis, indicating that loss of PTRN-1 function in the larval epidermis is required for suppression of the dapk-1(ju4) phenotype.To identify new e allele   . Suppresdhc-1(ju697), which results in a missense change G2537S in the fifth P-loop of DHC-1. Complete loss of function in dhc-1 causes lethality (dhc-1(or195ts) fully suppressed dapk-1(ju4) morphological defects at 20°C (dhc-1(or195) or dhc-1(or195); ptrn-1(0) caused more complete suppression of dapk-1(ju4) phenotypes than did ptrn-1(0), suggesting DHC-1 might affect additional pathways required for dapk-1 function.Our screen also identified a mutation in the dynein heavy chain, ethality ; an inde at 20°C . This su at 20°C . PTRN-1  at 20°C , and dynju1143 and ju1145, which result in nonsense mutations (Q608stop and R48stop) in dapk-1(ju4) (dapk-1(ju4) Mor phenotypes from 100% to 40% and 30% respectively, and resemble the dapk-1(gk219) deletion allele and other newly generated deletions of the dapk-1 locus (dapk-1(ju4) phenotypes are recessive, the identification of such intragenic suppressors suggests ju4 causes a gain of function (see Discussion). Both ptrn-1(lt1) and dhc-1(or195) completely suppressed dapk-1(gk219) morphological phenotypes  . These m-1 locus . Althougenotypes , suggestunc-116, which encodes a kinesin-1 plus-end directed motor, suppressed dapk-1(ju4) morphological defects. DAPK family members have previously been implicated in MT stability, promoting function of the MT-associated protein tau via the Pin1 prolyl isomerase or the MARK kinase  did not modify dapk-1(ju4) phenotypes , both in ptrn-1(+) and in ptrn-1(0) backgrounds (dapk-1(gk219) Mor phenotypes. None of these mutants conferred morphological defects in a dapk-1(+) background  defects were not suppressed by loss of function in several other genes implicated in MT dynamics  in the epidermis (dapk-1(ju4) animals constitutively express high levels of AMPs such as nlp-29 and nlp-30, and this hyperactive epidermal immune response is genetically separable from dapk-1(ju4) epidermal morphological defects  and unc-116(e2310). Neither mutant suppressed the elevated innate immune response in dapk-1(ju4)  fully suppressed the elevated immune response of dapk-1(ju4)  , as meask-1(ju4) . Among aptrn-1(0) mutants, PTRN-1 acts redundantly with γ-tubulin and the ninein-like protein NOCA-1 to assemble noncentrosomal MT arrays essential for epidermal development (dapk-1(ju4) epidermal defects  single mutants displayed significantly delayed wound closure compared to the wild type. Thus, DAPK-1 and PTRN-1 play antagonistic roles in epidermal development and in wound repair.e wounds . We founpe rates . Moreovedapk-1 epidermal phenotypes are suppressed by loss of function in a MT stabilizing factor (Patronin/ptrn-1) and enhanced by loss of function in MT destabilizing factors  we hypothesized that epidermal defects in dapk-1 mutants might result from excessive stabilization of epidermal MTs. We therefore tested whether drugs that depolymerize MTs  or stabilize MTs (paclitaxel) could modify epidermal Mor defects. Some experiments used cat-4 mutants, which are defective in biopterin synthesis and display leaky cuticles and hypersensitivity to drugs (Because the to drugs .dapk-1(gk219) animals (cat-4 itself strongly enhanced dapk-1(gk219), suggesting that dapk-1 is also sensitized to defects in cuticle integrity (dapk-1(gk219) cat-4 animals were suppressed by colchicine in a dose-dependent manner, and high concentrations of colchicine (5 mM) significantly suppressed dapk-1(ju4) phenotypes (dapk-1(gk219) morphological defects, consistent with Mor phenotypes in dapk-1(ju4) mutants being caused by hyper-stabilized MTs  mutants display overtly normal epidermal morphology, but were six times more sensitive to the effects of paclitaxel compared to WT animals (dapk-1(gk219) ptrn-1(0) double mutants (dapk-1(gk219) single mutants (dhc-1(or195) single mutants were also hypersensitive to paclitaxel, compared to wild type  innate immune responses. Conversely, 5 μM paclitaxel induced Pnlp-29-GFP expression in 40% of ptrn-1(0) mutants, including in animals not displaying strong Mor phenotype  mutants, focusing on the anterior epidermis, where dapk-1 morphological defects begin. In early (L2) larvae the MT architecture of dapk-1(ju4) animals appeared normal (dapk-1(ju4) animals (dapk-1(ju4) adults MT bundles were spaced normally (dapk-1(ju4) animals MT bundles were straighter and more bundled than in wild type (dapk-1(ju4) mutants displayed increased MT bundle anisotropy compared to wild type (dapk-1(gk219) mutants were very similar to those of dapk-1(ju4)  . The simdapk-1(ju4). ptrn-1(0) mutant larvae have slightly fewer circumferential MT bundles (ptrn-1(0) adults displayed a significant loss of circumferential MT bundles  adults displayed normal anisotropy (dapk-1(ju4) ptrn-1(0) double mutants the number and length of circumferential MT bundles resembled that of ptrn-1(0) mutants (ptrn-1(0) suppressed the increased anisotropy of lateral MTs in dapk-1(ju4) to wild type levels . EBP-GFP binds to growing MT plus ends and in vivo forms moving comets whose movement can be quantified by kymograph analysis (see Methods). We found that MT plus ends in the wild type adult epidermis were highly dynamic, with 0.09 ± 0.015 comets/μm2 in the lateral epidermis, and slightly more (0.12 ± 0.015 comets/μm2) in the dorsoventral epidermis (ptrn-1(0) animals was similar to that in wild type (dapk-1(ju4) mutants, as well as dapk-1(ju4) ptrn-1(0) double mutants, had significantly more comets in the lateral epidermis than in WT or ptrn-1(0) (dapk-1(ju4) mutants, and increased in ptrn-1(0) single and in dapk-1(ju4) ptrn-1(0) mutants (dapk-1(ju4) mutants thus display more slow-growing MT plus ends in the lateral epidermis, consistent with a partial stabilization of MT dynamics.We next assessed the effects of pidermis ,2. The oild type , as previld type . By conttrn-1(0) ,4. In witrn-1(0) . MT grow mutants . dapk-1(ptrn-1(0) mutant and dapk-1(ju4) ptrn-1(0) backgrounds. However, dapk-1(ju4) mutants displayed a significant bias in comet directionality in the dorsoventral epidermis, such that fewer comets grew away from the lateral epidermis. Thus, in dapk-1 mutants growing MTs are more confined to the lateral epidermis and less likely to extend into the dorsoventral epidermis.Finally, we analyzed the directionality of plus-end growth. In the lateral epidermis most EBP-GFP comets grew anteriorly or posteriorly, without a strong bias in directionality. Similarly, in the dorsoventral epidermis equal numbers of EBP-GFP comets grew towards or away from lateral epidermal ridges . EBP-GFPdapk-1 mutant we tested individual PTRN-1 domains. Like other CAMSAP proteins, PTRN-1 has three conserved regions: an N-terminal calponin homology (CH) domain, of unknown function; a central coiled-coil (CC) domain, known to interact with cytoskeleton associated proteins, and a C-terminal MT-binding CKK domain specific to CAMSAPs (dapk-1(ju4) ptrn-1(0) mutants as multicopy transgenes using the dpy-7 promoter. As shown above, expression of full-length PTRN-1 rescued the ptrn-1(0) suppression phenotypes  ptrn-1(0) double mutants, whereas constructs lacking the CKK domain could not rescue, suggesting the CKK domain is required for PTRN-1 function  mutants caused highly penetrant Mor phenotypes, resembling those of dapk-1(ju4) (ptrn-1(0) mutants did not induce Mor phenotypes (not shown). Paralleling our rescue analysis, transgenes expressing the CKK domain and the CH or CC domains did not cause epidermal defects in ptrn-1(0) animals. Moreover, expression of the CKK domain only caused aberrant development in a ptrn-1(0) background and not in a wild type background (ptrn-1(0) mutants induced Pnlp-29-GFP expression, similar to that seen in dapk-1(ju4) mutants  . This isckground , suggest mutants , and acc mutants . These dIn other CAMSAP proteins the CKK domain binds MTs , whereasThe PTRN-1 CH domain did not confer subcellular localization, whereas fragments lacking the CH domain localized to filaments and puncta, resembling full length PTRN-1 , Table 2ptrn-1(0) mutants (ptrn-1(0) background caused circumferential MT bundles to be straighter than in wild type, resembling the effects of paclitaxel treatment (ptrn-1(0) mutants expressing full-length PTRN-1 did not display straighter MT bundles or Mor phenotypes  mutants, PTRN-1’s MT stabilization activity, mediated by its CKK domain, might be aberrantly active. We therefore assessed whether PTRN-1 localization or expression was altered in dapk-1(ju4) mutants. In the wild type, PTRN-1::GFP formed thick filaments or puncta in the anterior lateral epidermis, and sparse filaments in the body epidermis (dapk-1(ju4) adults, PTRN-1::GFP formed twice as many thick filaments in the head (dapk-1(ju4), as multicopy GFP::PTRN-1 transgenes (dapk-1(ju4) mutants compared to WT  and dapk-1(ju4) ptrn-1(0) double mutants caused increased GFP::PTRN-1 filament formation (dapk-1(ju4) could reflect a positive feedback loop whereby stabilized MTs recruit more PTRN-1, resulting in further MT stabilization.Increased PTRN-1 filaments in ormation . The incTo address how DAPK-1 might regulate PTRN-1 we examined whether DAPK-1 was capable of physical association with PTRN-1. Using co-immunoprecipitation in transfected HEK293T cells we found that PTRN-1 can be pulled down with DAPK-1 , suggestdapk-1 mutant defect in epidermal morphology suggests DAPK-1 might inhibit MT stability, possibly via negative regulation of PTRN-1 itself. DAPK-1 might act directly or indirectly on PTRN-1. Although DAPK family members have been shown to localize to the cytoskeleton, the subcellular location of DAPK relative to MTs or PTRN-1 has not been examined. To further address whether DAPK-1 might affect PTRN-1 or MTs directly, we examined the localization of functional GFP::DAPK-1. Unexpectedly, GFP::DAPK-1 formed discrete puncta that underwent rapid, directed movement within the larval or adult epidermis  nor did they induce dapk-1-like phenotypes (dapk-1(ju469) (ju4 mutant background (dapk-1 phenotypes suggests DAPK-1(ΔKinase) inhibits DAPK-1 in a dominant-negative manner and may also inhibit parallel pathways.We asked whether DAPK-1’s transport correlated with its functions in epidermal morphology. DAPK-1, like mammalian DAPK, contains an N-terminal kinase domain, a Calcium/Calmodulin binding domain, a set of ankyrin repeats, a P-loop, a cytoskeleton-interacting domain and a C-terminal death domain  Figure . Construenotypes , Table 41(ju469) , and cauWe examined the localization and dynamics of these truncated proteins. Both GFP::DAPK-1(ΔKinase) and GFP::DAPK-1(S179L) formed dynamic puncta that moved at normal or slightly slower velocities . Conversdapk-1 suppressors. The velocity of DAPK-1 puncta did not change in unc-116/kinesin-1 mutants, but decreased two-fold in dhc-1(or195) mutants (ptrn-1(0) mutants had fewer moving DAPK-1 puncta in the dorsoventral epidermis, consistent with their reduced numbers of circumferential MTs (ptrn-1(0) nor dhc-1 mutants affected the directional bias of GFP::DAPK-1 transport  allele, which results in a missense S179L alteration in the peptide-binding ledge of the DAPK-1 kinase domain. This allele, unlike dapk-1(gk219) or deletions of the dapk-1 locus, causes fully penetrant morphological phenotypes. Our identification of stop codon mutations as intragenic revertants, as well as the finding that transgenic expression of S179L DAPK-1 induces Mor phenotypes, suggests dapk-1(ju4) is a gain of function mutation.We screened for suppression of morphological defects of the dapk-1(ju4) has a more penetrant effect on epidermal morphology than the complete loss of dapk-1 function is that the S179L change alters the phosphorylation specificity of DAPK-1. Like mammalian DAPK family members, DAPK-1 likely has many phosphorylation targets, some of which may affect epidermal morphology. One clue to the effect of S179L on protein function comes from our structure function study. Truncated versions of mammalian DAPK1 can have dominant-negative behavior  did neither. Thus, DAPK-1(S179L) is unlikely to be kinase-dead, but may have an aberrantly regulated kinase activity. Inappropriate activity of DAPK-1 targets in dapk-1(ju4) mutants may account for the stronger morphological defects in this genetic background.How the S179L change causes a gain of function remains to be determined. One hypothesis for why behavior . In our ptrn-1 and dapk-1; thus, DAPK-1 may be regulating epidermal MT stability at least partially through PTRN-1. In addition, we identified dhc-1 and other cytoskeletal associated proteins that specifically modify dapk-1 morphological phenotypes; such genes may act downstream of DAPK-1.Our pharmacological data suggest DAPK-1’s normal function is to restrain epidermal MT stabilization. Our suppressor screen and tests of candidate genes suggests a specific interaction between dapk-1(ju4) is genetically separable from epidermal morphology defects  single mutant larvae show grossly wild-type epidermal MT plus-end dynamics and have slightly fewer circumferential MT bundles in the epidermis  suppression phenotypes, but can induce epidermal defects when overexpressed. Depending on the particular CAMSAP family member, the CKK domain can target MT minus ends, or can bind along the lattice  results in aberrant MT stabilization. DAPK-1 might inhibit the activity of the CKK domain indirectly via the CC or CH domains .10.7554/C. elegans epidermis, and why aberrant DAPK-1 function should trigger altered epidermal morphology.To our knowledge, DAPK family members have not been previously shown to undergo MT-dependent transport. Many questions remain concerning the mechanism of DAPK-1 transport; here we speculate on the possible function of DAPK-1 motility in the C. elegans epidermis the MT cytoskeleton is required for cell shape changes  were scored in animals grown at 20°C unless stated otherwise. Strains were constructed using standard procedures, and were genotyped by PCR or sequencing. Previously described mutations include: dapk-1, ptrn-1. New dapk-1 suppressor mutations and additional candidate genes tested are listed in dapk-1 CRISPR deletions were generated by injecting 27 µM circular RNAs (crRNA) CACCATTAACATTCTCTCTC and ATACGAACGCATAATAA, dpy-10 crRNA as a co-injection marker, tracrRNA, and purified Cas9 protein , following published methods  animals with 47 mM EMS using standard protocols and screened the F2 progeny (~5000 haploid genomes) for suppression of Mor phenotypes. dapk-1(ju4) suppressors were outcrossed once prior to extraction of genomic DNA and whole genome sequencing (BGI Americas). Sequence data were processed and analyzed using MAQgene  or a Galaxy workflow, comparing datasets to each other and to the wild type reference sequence in WS220. We used SNP mapping to locate suppressor ju698 to a 2.2 Mb interval within which we identified a stop codon mutation in ptrn-1. We mapped ju697 to chromosome I, where we identified a missense alteration in dhc-1.We mutagenized E. coli OP50. Two days later, the embryos were plated and allowed to develop at 20°C. Three days later, young adult animals were scored for epidermal defects or assayed for antimicrobial reporter gene expression. To visualize MTs after drug treatment, L4 animals were anesthetized in 0.5 mM levamisole and 2 μM paclitaxel or 2 mM colchicine solution for 2 hr before imaging.Solutions of drugs in M9 were pipetted onto small NGM agar plates . Plates were dried for 24 hr, then seeded with Needle wounding and actin ring quantitation were performed as described .C. elegans for imaging, including muscimol, 1-phenoxy-2-propanol, and levamisole. GFP::DAPK-1 dynamics were also highly sensitive to physical stress; for example, immobilization in 10% agarose and 0.05 μm beads  in the lateral epidermis or a 300 μm2 ROI in the dorsoventral epidermis.We imaged subcellular dynamics using a custom-built microscope (Solamere Technologies) with a Yokogawa CSU-X spinning disk confocal head, a Photometrics Cascade II EM-CCD camera, and a Zeiss NA 1.46 objective. Images were acquired at 4.2 Hz, with exposure time 114 ms at 1000 x magnification, for 100 or 200 frames. We generated kymographs using Metamorph software and determined particle velocity by manual measurement of track angles. We manually counted moving puncta or comets in a 10 frame window, using a 200 μmWorm protein samples were prepared by boiling 10 μl pellets of mixed-stage worms in SDS-mercaptoethanol solution. We detected PTRN-1 using antibody OD208A ; we used2) for 30 min at 4°C. M2-FLAG conjugated magnetic beads (Sigma) were used for IP, and mouse anti FLAG  and anti HA  antibodies were used for western blotting. The co-IP was repeated twice.To test co-immunoprecipitation, a 1:1 ratio of tagged DAPK-1 and PTRN-1 were co-transfected using Lipofectamine 2000 (Invitrogen) into HEK293 cells growing on poly-D-Lysine-coated plates (Sigma-Aldrich) in Opti-MEM (Thermo Fisher Scientific). After two days, cells were collected in cold PBS and lysed in lysis buffer  expression we took images on a Zeiss compound microscope using a GFP long-pass filter set. To quantify Pnlp-29-GFP (frIs7) expression levels we used the COPAS C. elegans BIOSORT  at the Troemel lab (UCSD). We calculated the ratio of green fluorescence to the internal control Pcol-12-dsRed.For visual comparisons of Pcol-19-GFP::TBB-2, juSi239): (1) We calculated the anisotropy index using the ImageJ plug-in FibrilTool  We measured MT bundle length by drawing a line scan along a circumferential MT, from the edge of the lateral epidermis or the dorsal or ventral midline, to the end of the bundle. MT bundles detached from the epidermal ridges were not measured. (3) We counted MT bundles in a 40 μm ROI along the lateral epidermis along the anteroposterior axis. (4) We counted MT bundles crossing a 40 μm line in the dorsoventral region, including bundles not attached to the lateral or dorsoventral networks. For parameters 2–4 the ROI begins 40 μm behind the nose tip and extends 40 μm posteriorly. Schematics of ROIs are in Quantitation of MT bundles : (1) Number of filaments in the dorsoventral regions of the head were counted in an ROI extending 40 μm from the nose tip  Filaments were counted in the anterior lateral epidermis in a 200 μm2 ROI. A line of GFP > 1 μm was considered a filament. We did not examine the defective region to avoid quantifying changes in localization secondary to the aberrant epidermal morphology.Quantitation of GFP::PTRN-1 filaments (Pnose tip . (2) FilAll statistical analyses were performed with GraphPad Prism. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Fiona Watt as the Senior Editor and four reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your article "DAPK interacts with Patronin and the microtubule cytoskeleton in epidermal development and wound repair" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. We hope you will be able to submit the revised version within two months, so please let us know if you have any questions first.Summary:C. elegans epidermal cells as a model. Through a suppressor screen, they uncover that DAPK-1 functions through the microtubule cytoskeleton and more specifically the minus end binding protein patronin. The authors present rigorous genetic and pharmacological evidence suggesting that the epidermal defects associated with aberrant DAPK-1 function are due to hyperstabilization of the microtubule cytoskeleton caused by misregulation of patronin.In this report, Chuang et al. explore the role of the death associated protein DAPK-1, a structurally unique 160-kD calmodulin dependent serine-threonine kinase that has been implicated in a range of processes in mammals, using Essential revisions:ju4 (gain of function) and gk219 (loss of function) alleles are rescued by mutations in patronin and dhc. The interchangeable use of these alleles (and their ability to act similarly) is not well explained. Does the deletion mutant have the same microtubule morphology defects as the gof? It is necessary to fill in the gaps in experiments using gk219 for all experiments, as the nature of this allele is understood.Both the Of course, if you have data on physical interaction between DAPK and Patronin, these would greatly help the paper. Both the ju4 (gain of function) and gk219 (loss of function) alleles are rescued by mutations in patronin and dhc. The interchangeable use of these alleles (and their ability to act similarly) is not well explained. Does the deletion mutant have the same microtubule morphology defects as the gof? It is necessary to fill in the gaps in experiments using gk219 for all experiments, as the nature of this allele is understood.ju4, which displays 100% penetrance of the Mor phenotype and is easiest to score, and gk219, which has partial penetrance, allowing us to survey for enhancement as well as suppression of the phenotype. We believe gk219 represents the null phenotype of dapk-1, as explained below. The ju4 missense alteration is a gain of function that appears to have dominant-negative activity, as the phenotypes overall resembles a more penetrant version of the null phenotype.We use two different alleles, gk219 allele. We examined MT arrays in gk219 and in gk219 ptrn-1 mutants, and show the data in ju4 and ju4 ptrn-1 mutants. We had previously performed pharmacological tests on gk219, as shown in dapk-1 alleles. We now have also added the wound closure data on gk219 and gk219 ptrn-1 mutants. Overall, the morphological, cytoskeletal and wound healing defects of gk219 mutants are either similar to or slightly weaker than those of dapk-1(ju4).As requested, we have repeated some key experiments with the dapk-1(gk219) deletion allele, we used CRISPR/Cas9 to generate deletions of the entire dapk-1 transcription unit (23 kb)  . Three sdapk-1(ju4) mutation by structure-function analysis, as shown in dapk-1(ju4)/DAPK-1(S179L). We speculate that the S179L alters how the DAPK-1 kinase interacts with substrates and have expanded our discussion of this point .We explored the nature of the Of course, if you have data on physical interaction between DAPK and Patronin, these would greatly help the paper.To address this point we have now performed co-immunoprecipitation experiments in HEK293T cells. As shown in


Here, we announce the complete genome sequence of the earliest d-tartrate-fermenting Salmonella enterica subsp. enterica serovar Paratyphi B isolate harboring mcr-1 from the collection of the German National Reference Laboratory for Salmonella.In 2015, plasmid-mediated colistin resistance was reported to be caused by a mobilized phosphoethanolamine transferase gene ( Since tar Java) . MultidrL) genes , 4. In 2om China  and lateom China . These fS. Paratyphi B (dTa+) acquired the mcr-1 gene in the year 2008. Our earliest observed mcr-1-positive isolate (08-00436) originated from chicken skin. PCR screening of isolates from the collection of the German National Reference Laboratory for Salmonella revealed that de novo genome assembly based on 55,146 reads from a single-molecule real-time (SMRT) cell  was performed using the SMRT Analysis (version 2.3.0) software , resulting in 3 contigs with an average coverage of 143.12× per consensus base. Short-read sequencing using Illumina MiSeq technology was carried out on the same DNA sample. Paired reads were mapped against PacBio contigs using CLC Genomics Workbench 9.5.2. The contigs were closed to circular molecules, and errors in homopolymeric regions were corrected. The resulting genome contains a chromosome  and two plasmids, pSE08-00436-1  and pSE08-00436-2,  comprising an average G+C content of 52%. A single colony of isolate 08-00436 was picked and cultured for 16 h at 37°C in lysogeny broth supplemented with 2 mg/liter colistin-sulfate . Genomic DNA was extracted using the PureLink genomic DNA minikit . Genome sequencing was performed by the GATC Biotech AG  using the PacBio RSII system. A http://www.genomicepidemiology.org) revealed that the isolate belonged to the multilocus sequence type (MLST) 28 (ST28) and carries two copies of the class 2 integron, as well as additional resistance genes in the chromosome. Further resistance genes, including mcr-1, were located on the multireplicon IncHI2/IncHI2A/IncQ1 plasmid pSE08-00436-1. Initial genome annotation was performed using the automated Prokaryotic Genome Annotation Pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). Analysis of the results revealed 5,071 coding sequences (CDSs) , as well as 117 RNA genes . Further assessment of the genes involved in resistance, plasmid transfer, and virulence is in progress.Genome analysis using tools of the Center for Genomic Epidemiology (CP020492 (chromosome), CP020493 (pSE08-00436-1), and CP020494 (pSE08-00436-2).Sequences were deposited in GenBank under the accession numbers