Mycobacterium tuberculosis Rv0652, as an immunoadjuvant for DC-based tumor immunotherapy, and its capacity for inducing DC maturation was investigated. In this study, we showed that Rv0652 is recognized by Toll-like receptor 4 (TLR4) to induce DC maturation, and pro-inflammatory cytokine production  that is partially modulated by both MyD88 and TRIF signaling pathways. Rv0652-activated DCs could activate naïve T cells, effectively polarize CD4+ and CD8+ T cells to secrete IFN-gamma, and induce T cell-mediated-cytotoxicity. Immunization of mice with Rv0652-stimulated ovalbumin (OVA)-pulsed DCs resulted in induction of a potent OVA-specific CD8+ T cell response, slowed tumor growth, and promoted long-term survival in a murine OVA-expressing E.G7 thymoma model. These findings suggest that Rv0652 enhances the polarization of T effector cells toward a Th1 phenotype through DC maturation, and that Rv0652 may be an effective adjuvant for enhancing the therapeutic response to DC-based tumor immunotherapy.A key factor in dendritic cell (DC)-based tumor immunotherapy is the identification of an immunoadjuvant capable of inducing DC maturation to enhance cellular immunity. The efficacy of a 50S ribosomal protein L7/L12 (rplL) from  Because DC maturation is critical for the generation of effective immunity in therapeutic studies Dendritic cells (DCs) are the most important regulators of naïve cells during the generation of T cell-mediated immune responses, and DCs are the most potent antigen-presenting cells (APCs) invitroThe use of Toll-like receptor (TLR)-mediated DC activation may be an effective cancer treatment strategy because TLR activation usually induces Th1 responses Mycobacterium and related genera, and these adjuvants are predicted to be crucial components of immunotherapeutic strategies Mycobacterium tuberculosis ligands such as PE-PGRS and LprA are known to interact with and activate APCs, specifically DCs M. tuberculosis heparin-binding hemagglutinin (HBHA) is a potent immune adjuvant with potential applications in antitumor immunotherapy Proteins that polarize the activated immune systems toward a Th1 response are expressed by specific bacteria of the genus M. tuberculosis protein Rv0652, the 50S ribosomal protein (rp) L7/L12 (rplL) M. tuberculosis Rv0652 in DCs and their potential as adjuvants in DC-based antitumor immunotherapy are described. Rv0652 is shown to be a potent TLR4 agonist, inducing the MyD88 and TRIF signaling pathways, and possibly enhancing DC activation and Th1 polarization. Notably, Rv0652 mediated a strong induction of Ag-specific, CD8+ class I-restricted, CTL responses, and E.G7 tumor regression invivo. These results highlight the potential use of Rv0652 for DC-based antitumor immunotherapy.The biological activity and cellular immunity of b and I-Ab) mice were purchased from the Korean Institute of Chemistry Technology . C57BL/6 OT-I and C57BL/6 OT-II T-cell receptor transgenic mice, C57BL/6J TLR2 knockout mice , C57BL/10 TLR4 knockout mice , MyD88 knockout mice, and TRIF-deficient mice aged 6–8 weeks were purchased from Jackson Laboratory . Mice were housed in a specific pathogen-free environment within an animal facility for at least 1 week before the experiment. All animal procedures were performed in accordance with the protocols approved by the Konkuk University Institutional Animal Care and Committee and the recommendations for the proper use and care of laboratory animals (IACUC number: KU13047).Male 6 to 8-week-old C57BL/6 (H-2Kb and I-Ab) mouse, and E.G7, an ovalbumin (OVA)-expressing EL4 variant, were purchased from the American Type Culture Collection  and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 ug/ml streptomycin, and 10 mM l-glutamine  at 37°C in 5% CO2.EL4, a thymoma-derived cell line from the C57/BL/6 (H-2KEscherichia coli O111: B4) was purchased from Invivogen . H-2Kb-restricted OVA peptide (OVA257–264) and H-2b-restricted OVA peptide (OVA323–339) were synthesized by Peptron . The following FITC- or PE-conjugated monoclonal Abs were purchased from BD Biosciences : CD11c (HL3), CD80 (16-10A1), CD86 (GL1), Iab beta-chain (AF-120.1), H-2Kb (AF6-88.5), CCR7 (CD197), IL-10 (JESS-16E3), and IL-12p40/p70 (C15.6). Alexa568 conjugated anti-mouse IgG antibodies were purchased from Invitrogen . FITC-conjugated Mouse IgG Abs and cytokine ELISA kits for murine IL-1beta, IL-2, IL-6, IL-10, IL-12p70, TNF-alpha, and IFN-gamma were purchased from eBiosciences .Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, CCL19, and the FITC-annexin V/propidium iodide kit were purchased from R&D Systems . Dextran-FITC  was purchased from Sigma-Aldrich . Lipopolysaccharide . Briefly, rplL encoding Rv0652 was amplified by polymerase chain reaction (PCR) using M. tuberculosis H37Rv genomic DNA (ATCC27294) as template and the following primers: forward, GGA TCC ATG GCA AAG CTC TCC-3′5′-GGG CCC ; and reverse GAA TTC CTT GAC GGT GAC GGT-3′5′-GGG CCC . The PCR product was cut with BamHI and EcoRI and then inserted into pET28a vector cut with the same restriction enzymes. The recombinant plasmid was transformed into E. coli BL21 cells carrying bacteriophage DE3 for protein overexpression.Recombinant Rv0652 (rRv0652) protein was produced using pET28a vector  and an DCs were isolated and cultured as recently described 6 cells), followed by tail vein injection of immature and Rv0652-treated DCs (1×106 cells), some of which were pulsed with OVA (1 ug/ml) on days 1, 3, and 5 after tumor inoculation. Groups of tumor-bearing mice were treated with PBS, iDCs (immature DCs), DCs-OVA (DCs pulsed with OVA peptide), LPS-DCs-OVA (LPS-treated DCs pulsed with OVA peptide), or Rv0652-DCs-OVA (Rv0652-treated DCs pulsed with OVA peptide). Tumor size was measured every 2 days, and tumor mass was calculated as: V = (2A×B)/2, where A is the length of the short axis and B is the length of the long axis.Mice were injected subcutaneously (s.c.) into the right lower back with EL4 or E.G7 thymoma cells , then stained with anti-mouse Rv0652 and Alexa568 conjugated anti-mouse IgG antibodies overnight at 4°C to detect Rv0652. Fluorescence intensity was analyzed using a confocal laser scanning microscope . Images were acquired using LSM510 Meta software and processed using the LSM image examiner.invitro and invivo chemotaxis were performed as previously described (26).Antigen uptake and 257–264 (0.5 ug/ml) or OVA323–339 (0.5 ug/ml) for 24 h. CFSE-labeled OT-I and OT-II T-cells were seeded in triplicate wells at a density of 1×105 cells per well, together with DCs at a density of 1×104 cells per well, in U-bottomed 96-well microtiter culture plates (Nunc). Cells were harvested after 96 h, stained with Cy5-labeled anti-CD8 and anti-CD4 monoclonal Abs, and analyzed by flow cytometry (BD Biosciences).In brief, DCs were incubated with Rv0652 or LPS in the presence or absence of OVA257–264 (DC-OVA), or Rv0652-treated DCs pulsed with OVA257–264 (Rv0652-DC-OVA) on days 1 and 7. Spleen cells (2×106 per well) from injected mice (day 10) were restimulated with 10 ug/ml OVA257–264 for 7 days in 6-well plates, then co-cultured with EL4  and E.G7  cells. After 2 days, mixed cells were analyzed for cytotoxicity against tumor cells by flow cytometry. After 1 day, the culture supernatant was used for IFN-gamma and IL-2 ELISAs.BALB/c mice were injected with PBS, untreated DCs (iDC), DCs pulsed with OVAt-test. Differences with a p-values of <0.05 were considered statistically significant. Datasets of survival curves were analyzed using the Kaplan–Meyer log-rank test. Statistical analyses were conducted using GraphPad Prism 4 software.Statistical significance was assessed using the Student’s paired −/−, or TLR4−/− mice were stimulated with Rv0652 (0.5 ug/ml), and Rv0652 was detected on the cell surface with Alexa568-conjugated anti-Rv0652 monoclonal antibodies. Rv0652 bound to the surfaces of WT and TLR2−/− DCs, but not TLR4−/− DCs, , we confirmed that endotoxin contamination of rRv0652 did not occur (<15 pg/ml) (Data not shown). As shown in Soluble rRv0652 was carefully purified from in Next, we examined the effect of rRv0652 on the viability of DCs by performing FACS analysis of PI/Annexin V stained cells. As shown in −/− and TRIF-deficient mice were compared. The rRv0652-induced production of IL-1beta, IL-6, and TNF-alpha was partially inhibited in the absence of MyD88 and TRIF  with Rv0652-DCs pulsed with OVA257–264 (323–339 (+ or CD8+ T cells (CFSE-labeled and OVA-specific) with DCs that were pulsed with OVA257–264 or OVA323–339, but not stimulated with rRv0652. Syngeneic CD4+ and CD8+ T cells primed with Rv0652-DCs produced significantly (P≤0.05–0.01) higher levels of IFN-gamma production than those primed with iDCs  transgenic CD8A257–264  or OVA32(323–339  induced ith iDCs . These r257–264 (OVA-DCs) and injected intravenously resulted in a significantly slower growth of E.G7 tumors than intravenous injection of PBS or iDCs (257–264 (Rv0652-OVA-DCs) and mice that received LPS-DCs pulsed with OVA257–264 (LPS-OVA-DCs) as positive control were significantly smaller than E.G7 tumors in mice that received OVA-DCs  significantly elevated Ag-specific CTL antitumor activity against an E.G7-OVA expressing thymoma.We showed that rRv0652 could induce antigen specific antitumor effects by triggering DC maturation. Rv0652 improved the efficacy of DC-based antitumor immunotherapy in mice by acting as a potent adjuvant. Evidence for these novel characteristics of rRv0652 include: 1) increased expression of surface molecules (CD80/CD86); 2) involvement of the MyD88- and TRIF-pathways in the production of pro-inflammatory cytokines induced by Rv0652 in DCs; 3) DC activation was not due to endotoxin contamination and occurred via TLR4 but not TLR2 signaling; 4) there is an increased migratory capacity in Rv0652-DCs; 5) the effect of Rv0652-DCs on the expansion of IFN-gamma-producing CD4+ and cytotoxic CD8+ T cells. We found that Rv0652-DCs significantly increased their surface molecules and the production of IL-12p70, which is essential for Th1 polarization + and CD8+ T-cells. The production of IFN-gamma is an important feature of Th1 immune responses, and this result suggests that Rv0652 may promote a Th1 immune response through the activation and maturation of DCs. Furthermore, Rv0652 protein enhanced CCR7 expression, which is crucial for DC migration from the periphery into the paracortex of secondary lymphoid organs invitro and to draining LNs invivo. Therefore, Rv0652 affects DC function in multiple ways, and Rv0652 may be an effective adjuvant for DC-based antitumor immunotherapy.Co-stimulatory signals, such as CD40, CD80, and CD86 as well as cytokines including IL12p70 from Ag-specific and activated DCs are required for the induction of proper host immunity mediated by helper CD4TLRs are expressed by numerous immune cells and play key roles in innate and adaptive immune responses M. tuberculosis ligands, such as LprA M. tuberculosis HBHA is a novel TLR4 agonist for antitumor vaccinations M. tuberculosis Rv0652, similar to HBHA, directly binds TLR4 and activates TLR4 signaling, leading to the expression of co-stimulatory and MHC antigen presentation molecules, as well as the production of IL-1beta by DCs. Kim et al−/− and TRIF−/− DCs still produce small amounts of pro-inflammatory cytokines. Further study is necessary to fully understand this process.Several purified +CD69+ T cells. Taken together, this increase in antitumor efficacy was attributed to the enhancement of Th1 polarization by Rv0652-DCsthat activate the CTLs that are able to lyse tumor cells. Our data indicate that M. tuberculosis Rv0652 can be used in DC-based antitumor immunotherapy.Systemic intravenous  and subcFigure S1Amino acid sequence of Rv0652 (www.tbdb.org).(TIF)Click here for additional data file.Figure S2257–264 protects mice against E.G7 tumor challenge.Rv0652-treated DCs pulsed with OVA C57BL/6 mice were challenged s.c with 3×105 E.G7 tumor cells into the right flank area. For administration of DCs (1×106 cells/mice), mice were injected subcutaneously with PBS, iDC, DCs pulsed with OVA257–264, or Rv0652-treated DCs pulsed with OVA257–264 on days 1, 3 and 5 after the tumor challenge. (A) Following tumor challenge with E.G7 tumor growth was monitored by measuring the diameter every 2 days. *P<0.05, each group, n = 10. (B) Survival of mice with E.G7 tumor challenge after injection of OVA peptide-pulsed and Rv0652-treated DCs, n = 10 mice/group. P value calculated by Kaplan-Meyer log-rank test between two groups of mice injected with iDC and Rv0652-DC-OVA, respectively.(TIF)Click here for additional data file.


We show that compromised APTF-2 activity leads to defects in dorsal intercalation, aberrant ventral enclosure and elongation defects, ultimately culminating in the formation of morphologically deformed larvae or complete arrest during epidermal morphogenesis. Using cell lineaging, we demonstrate that APTF-2 regulates the timing of cell division, primarily in ABarp, D and C cell lineages to control the number of neuroblasts, muscle and epidermal cells. Live imaging revealed nuclear enrichment of APTF-2 in lineages affected by the qm27 mutation preceding the relevant morphogenetic events. Finally, we found that another AP-2 TF, APTF-4, is also essential for epidermal morphogenesis, in a similar yet independent manner. Thus, our study provides novel insight on the cellular-level functions of an AP-2 transcription factor in development.The evolutionarily conserved family of AP-2 transcription factors (TF) regulates proliferation, differentiation, and apoptosis. Mutations in human AP-2 TF have been linked with bronchio-occular-facial syndrome and Char Syndrome, congenital birth defects characterized by craniofacial deformities and patent ductus arteriosus, respectively. How mutations in AP-2 TF cause the disease phenotypes is not well understood. Here, we characterize the  Caenorhabditis elegans, APTF-2, which falls in the same mutation hotspot as in human Char syndrome and Brancio-oculo-facial syndrome patients. Compromised APTF-2 activity in C. elegans results in embryonic lethality and embryos that survive to hatching displays body morphological defects, reminiscent of the aforementioned human diseases. Using time-lapse microscopy, we found that misregulation of cell division in the skin, muscle and neuronal cell lineages is the primary cause of developmental arrest. Our study provides insight into the regulation of cell division timing by AP-2 transcription factors and provides a model to study human diseases associated with AP-2 mutations.Mutations in the evolutionarily conserved family of AP-2 transcription factors are associated with multiple birth defects in Char syndrome and Brancio-oculo-facial syndrome. These DNA-binding proteins are known to regulate the proliferation, differentiation and death of specific cells during embryonic development but how point mutations in the AP-2 DNA-binding domain lead to these diseases during development is currently unknown. We have identified a mutation in one of the AP-2 orthologs of the nematode  For analysis using GFP reporters, F2 progeny exhibiting aptf-2 phenotypes and carrying the markers were selected from crosses between aptf-2(qm27) and the following strains: FT250 xnIs96 [pJN455(hmr-1p::hmr-1::GFP::unc-54 3'UTR) + unc-119(+)]  [otIs33[kal-1p::gfp] [zuIs178 [his-72(1kb 5' UTR)::his-72::SRPVAT::GFP::his-72 (1KB 3' UTR) + 5.7 kb XbaI—HindIII unc-119(+)]. stIs10024 [pie-1::H2B::GFP::pie-1 3' UTR + unc-119(+)] (a gift from Waterston lab) and JIM119 zuIs178 [his-72(1kb 5' UTR)::his-72::SRPVAT::mCherry::his-72 (1KB 3' UTR) + 5.7 kb XbaI—HindIII unc-119(+)]. stIs10024 [pie-1::H2B::mCherry::pie-1 3' UTR + unc-119(+)] (a gift from Waterston lab). die-1::gfp reporter strain was a gift from Hardin lab [Strains were maintained at 20°C under standard conditions . Wild-tyi et al.  and aptf-119(+)] , SU265 jsu1006)] , OH904 o1p::gfp] , RW10029aptf-2, the aptf-2 promoter (2 kb sequence upstream of aptf-2 start codon) followed by the aptf-2 coding sequence were amplified from N2 and aptf-2(qm27) animals, respectively and inserted into XbaI and AgeI sites upstream of gfp in the original pPD95.75 vector. The wild-type aptf-2-containing plasmid was injected into the gonad of aptf-2(qm27) hermaphrodite animals to examine its potency in rescuing aptf-2(qm27) phenotypes, whereas the plasmid containing mutated aptf-2 was injected into N2. This resulted in the following transgenes: msnEx15 ; aptf-2(qm27) and msnEx239 . Ten L4 larvae expressing wild-type aptf-2 were subjected to a UV source (BioRad) for 15 seconds to integrate the extrachromosomal array into the genome. Three hundred F2 worms were then singled and incubated for three weeks and subsequently examined for expression and embryonic lethality. Those expressing the transgene and giving rise to 100% viable progeny were selected and outcrossed. The resulting strain, RZB104 (aptf-2(qm27); msnIn104), was used throughout this study.To construct plasmids containing wild-type or mutated aptf-2 tagged with a nuclear localization signal (NLS), the amplified 4.3 kb genomic sequence containing the aptf-2 promoter and the coding region was inserted into XbaI and XmaI sites in pNL74.4 ). The transgenic animals were then crossed with aptf-2(qm27) or aptf-2(gk902) to assess the ability of NLS-tagged APTF-2 to rescue the aptf-2 mutants.To construct  pNL74.4 , a modifPvuII, 20 μg/μl rol-6(su1006) digested with SbfI and 5–10 μg/μl each construct digested with SbfI.Microinjection was performed as described by Mello and Fire . Injectimal-1(qm27) mutant worms using standard method and subjected to whole genome sequencing using Illumina platform and annotated using MAQGene  males was crossed with aptf-2(qm27) hermaphrodites. Non-GFP F1 animals were singled and incubated to lay embryos for 24 hours. The F1 animals were genotyped for the gk902 deletion and only the cross progeny between qm27 and gk902 alleles was scored for embryonic lethality of their F2s.For complementation assay, aptf-2(qm27) and aptf-2(gk902) were singled and incubated for 24 hours. Each animal was shifted to a new plate every day for 5 consecutive days to the point that no more embryos were laid. The total number of embryos laid was determined as the brood size. The number of hatched animals was calculated and used to determine the percentage of embryonic lethality. Larvae that did not grow into adult in 48–92 hours after hatching were considered as being arrested. aptf-2(qm27) and aptf-2(gk902) larvae of any stage were subjected to phenotypic analysis to determine the presence and the position of the morphological defects.For brood size analysis, ten L4 larvae of wild-type, aptf-2(qm27) and aptf-2(gk902) animals whose embryonic lethality was determined as described above, the embryonic lethality of the remaining strains were determined as follows: ten to fifteen gravid hermaphrodites were placed on the plate and incubated at 20°C for several hours to lay more than 100 embryos. Hermaphrodites were then removed and the number of embryos laid was counted. Twenty-four hours later, the number of larvae hatched was determined. Each experiment was repeated at least five times.Besides wild-type, 2 (Photometrics). All other images and movies were acquired using a spinning disk confocal system composed of a Nikon Ti Eclipse microscope with a CSU-X1 spinning disk confocal head (Yokogawa), DPSS-Laser (Roper Scientific) at 491 and 568 nm excitation wavelengths and an Evolve Rapid-Cal electron multiplying charged-coupled device camera (Photometrics). For both microscopes, Metamorph software (Molecular Devices) was used to control acquisition. Projected images were created using Fiji. All imaging was done at 20°C in an environmental chamber encompassing the microscope stage heated by a JCS temperature controller  within a microscope room kept at 18°C by a CITEC precision air conditioning unit.Larvae or embryos collected from gravid hermaphrodite were mounted onto 3% agarose-padded glass slide, closed with a coverslip and sealed with wax. DIC images shown in Figs aptf-4 dsRNA was synthesized as described [aptf-2(qm27) L4 larvae. Each animal was singled into a separate plate and its embryonic lethality was examined 24 hours post injection.escribed  and injeA. queenslandica (sponge), T. adhaerens (Placozoa), C. elegans (nematode), N. vectensis (sea anemone), D. melanogaster (fruit fly), S. purpuratus (sea urchin), C. intestinalis (tunicate), B. floridae (lancelete), D. rerio (fish), X. tropicalis (frog), G. gallus (chicken), H. sapiens (human). The resulting alignment was used to build and visualize a phylogenetic tree (neighbor-joining method) using Geneious (Biomatters Ltd.). Illustration of the gene and protein architecture was drawn using Illustrator for Biological Sequences [Protein sequence of the AP-2 transcription factor family members in the following metazoan species were aligned using Constraint-based Multiple Protein Alignment Tool (COBALT) : A. queeequences .Caenoharbditis species, and are located within the 3 kb region upstream of the start codon. Functional annotation was performed using DAVID Bioinformatics Resources 6.7 [AP-2 has been shown to bind to the palindromic consensus sequence 5'-GCCN3GGC-3', as well as the binding motif 5'-GCCN3/4GGG-3' . From thrces 6.7 , 46 and aptf-2(qm27) embryos expressing nuclear signal of GFP::HIS-72 and two embryos co-expressing APTF-2::GFP and mCherry::HIS-72 were analysed for at least 270 minutes according to the protocol described in [http://epic2.gs.washington.edu/Epic2.For cell lineaging, six ribed in –49. The ribed in  and compt-test was applied to compare the values.Statistical analyses were done using Prism 6 . Two-tailed Student’s S1 Figaptf-2(qm27) mutants visualized by DIC microscopy. (B) Gastrulation cleft closure in wild-type and aptf-2(qm27) embryos visualized by DIC microscopy. Ventral clefts are colored in blue and white arrows indicate ventral enclosure process. In the quantification of cleft size, error bar denotes mean ± s.e.m., p≤0.0001. (C) Ventral neuroblast migration to the midline in a wild-type embryo and their failure to migrate in an aptf-2(qm27) embryo. A maximum intensity projection of KAL-1::GFP expression is used to visualize neuroblasts. Red arrows indicate the progression of ventral cleft closure.(A) L4 of wild type and (TIF)Click here for additional data file.S2 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively. Defects in cell division are marked with an X.(TIF)Click here for additional data file.S3 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively.(TIF)Click here for additional data file.S4 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively.(TIF)Click here for additional data file.S5 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively.(TIF)Click here for additional data file.S6 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively.(TIF)Click here for additional data file.S7 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively. Defects in cell division are marked with an X.(TIF)Click here for additional data file.S8 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively.(TIF)Click here for additional data file.S9 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively.(TIF)Click here for additional data file.S10 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively.(TIF)Click here for additional data file.S11 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively.(TIF)Click here for additional data file.S12 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively. Defect in cell division is marked with an X.(TIF)Click here for additional data file.S13 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively. Defects in cell division are marked with an X.(TIF)Click here for additional data file.S14 Figaptf-2(qm27) embryos 1–3 are lineaged to 315 minutes, aptf-2(qm27) embryo 4 to 295 minutes, wild type, aptf-2(qm27) 5 and 6 are lineaged to 270 minutes respectively. Defects in cell division are marked with an X.(TIF)Click here for additional data file.S15 FigThe lineage of an embryo expressing APTF-2::GFP and HIS::mCherry was analyzed for nuclear enrichment of APTF-2::GFP. Nuclear enrichment is represented in red. APTF-2 is enriched in the AB and C lineages during embryogenesis at the time of ventral cleft closure and pre dorsal intercalation. The lineage was analysed to 232 minutes.(TIF)Click here for additional data file.S16 FigGeneious. Species: C. elegans (soil worm), A. queenslandica (sponge), T. adhaerens (a simple metazoan), N. vectensis (sea anemone), B. floridae (lancelet), D. melanogaster (fruit fly), C. intestinalis (tunicate), S. purpuratus (sea urchin), D. rerio (fish), G. gallus (chicken), X. tropicalis (frog), H. sapiens (human). C. elegans APTF-2, which is the subject of this study is highlighted in blue.The amino acid sequences were aligned using Constraint-based Multiple Protein Alignment Tool (COBALT) and the phylogenetic tree was built using neighbor-joining method and visualized using (TIF)Click here for additional data file.S17 FigBoth lineage trees shows enriched expression in the AB and C lineages while the MS, E and D has weak expression. Trees were drawn to 210 minutes.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S1 MovieMovie starts from early embryogenesis and ends at the time of hatching. Only a single focal plane of the embryo epidermis is shown. Time is indicated in minutes. Embryo at the bottom expresses APTF-2::GFP, whereas embryo at the top does not.(AVI)Click here for additional data file.S2 Movieaptf-2(qm27) embryo arrested during elongation. Time is indicated in minutes.Movie starts with two rows of dorsal epidermal cells ready to intercalate and ends with the wild-type embryo hatching and the (AVI)Click here for additional data file.S3 MovieMovie starts when ventral epidermal cells initiate embryo enclosure and ends with the wild-type embryo hatching, whereas the mutant embryo is not properly enclosed, resulting in the internal tissues leaking out from the gap on the ventral side. Time is indicated in minutes.(AVI)Click here for additional data file.S4 MovieMovie starts with two rows of dorsal epidermal cells ready to intercalate. The intercalation is abnormal and ends with the embryo being arrested during elongation. Time is indicated in minutes.(AVI)Click here for additional data file.S1 Text(XLSX)Click here for additional data file.S2 Text(ZIP)Click here for additional data file.S3 Text(ZIP)Click here for additional data file.S4 Text(ZIP)Click here for additional data file.S5 Text(ZIP)Click here for additional data file.S6 Text(ZIP)Click here for additional data file.S7 Text(ZIP)Click here for additional data file.


Macrophage proliferation and migration are important for many facets of immune response. Here we showed that stimulation of macrophages with type B CpG oligodeoxynucleotides (CpG-B ODNs) such as CpG-ODN 1668 increased the production of anti-inflammatory cytokine interleukin 1 receptor antagonist (IL-1Ra) in a TLR9- and MyD88-dependent manner. The CpG-B ODNs-induced IL-1Ra increased macrophage migration and promoted macrophage proliferation by down-regulating the expression of a cell cycle negative regulator, p27 to increase cell population in the S phase. The induction of IL-1Ra by CpG-B ODNs was F-spondin dependent. Knockdown of F-spondin and IL-1Ra decreased CpG-B ODNs-induced macrophage migration whereas overexpression of IL-1Ra increased migration of those cells. These findings demonstrated novel roles for F-spondin and IL-1Ra in CpG-B ODNs-mediated cell proliferation and migration of macrophages. Unmethylated CpG dinucleotides present in bacterial DNA are recognized by pattern recognition receptor Toll-like receptor 9 (TLR9) which triggers downstream signaling to activate target genes ,2. Like 1~42 peptide through maintaining IL-6 expression [in vivo [F-spondin is a secreted adhesion molecule that was originally isolated from the embryonic floor plate of vertebrates ,13, and pression . It has [in vivo . These s[in vivo . NeverthInterleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 type 1 receptor to block IL-1 signaling and elicits anti-inflammatory responses . In addiIn this study, we demonstrated that CpG-B ODNs, but not CpG-A ODNs, induced IL-1Ra expression in RAW 264.7 cells in a TLR9- and MyD88-dependent manner. The up-regulation of IL-1Ra in response to CpG-B ODN treatment was F-spondin dependent. The F-spondin/IL-1Ra signaling triggered by CpG-B ODN increased not only migration but also proliferation of macrophages. The effects of CpG-B ODN on the proliferation of macrophages were further explored by analyzing the cell cycle progression in the presence or absence of IL-1Ra overexpression.F-spondin and IL-1Ra genes were obtained from Santa Cruz Biotechnology . Anti-cyclin A antibody was purchased from GeneTex , anti-GAPDH was from Abcam , and anti-p21cip1 was from Upstate . Anti-F-spondin and anti-Myc antibodies were purchased from Biorbyt  and Abgent , respectively.CpG ODN1668 (5′-TCC ATG ACG TTC CTG ATG CT-3′), GpC ODN1668 (5′-TCC ATG AGC TTC CTG ATG CT-3′), CpG-ODN2006 (5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′) and Is ODN 6 (5′-GGG CAA CGT TCG ACG-3′) were synthesized with a phosphorothioate backbone at MDBio . CpG ODN 1585 (5′-GGG GTC AAC GTT GAG GGG GG-3′) and GpC ODN 1585 (5′-GGG GTC AAG CTT GAG GGG GG-3′) were purchased from InvivoGen . Lipofectamine 2000 and Amaxa cell line nucleofector kit V were obtained from Invitrogen  and Lonza , respectively. Chloroquine and was purchased from Sigma . Recombinant F-spondin proteins (rF-spondin), mouse IL-1Ra ELISA kit and anti-IL-1Ra antibody were purchased from R&D Systems . Anti-phospho CDK2 and anti-p27kip1 antibodies were obtained from Cell Signaling Technology . Anti-CDK2 and anti-actin antibodies as well as siRNAs for 2. THP1 cells were maintained in RPMI-1640 (Biowest) with the supplements described above and THP1-XBlue-defMyD cells were purchased from InvivoGen and cultured according to the manufacturer’s instructions.Cell lines used in this study were purchased from American Type Culture Collection unless otherwise specified. Murine macrophage cell lines RAW264.7 and J774A.1 were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum , 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified incubator at 37°C under 5% COBone marrow derived macrophages (BMMs) were harvested from BALB/c mice as described previously . In brieTotal RNA was extracted from cells using RNeasy kit  according to the manufacturer’s protocol. Amplification of specific target cDNA was performed using one microgram of total RNA and a one-step RT-PCR kit  which is designed to conduct the reverse transcription and PCR amplification in one tube from either total RNA or mRNA. The primers used were: forward 5′-ATG GAA ATC TGC TGG GGA CC-3′ and reverse 5′-CTA TTG GTC TTC CTG GAA GT-3′ for mouse IL-1Ra; forward 5′-GCC TCC GCA GTC ACC TAA TC-3′ and reverse 5′-CGC TGT CTG AGC GGA TGA AG-3′ for human IL-1Ra; forward 5′-AGC TTT CTC AGA TGA GAC CC-3′ and reverse 5′-CGT TTC TGT ACA ATG CTG GC-3′ for F-spondin; and forward 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′ and reverse 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′ for β-actin. PCR products were subjected to electrophoresis in a 1–2% agarose gel prepared with nucleic acid stain SafeView .For mouse F-spondin silencing, a target sequence 5′-TCC TAC TTC AGA GGT TTC ACG TTA A-3′ was used, and the control sequence was 5′-TCC TTC GAC GGA TTT CAC TGT ATA A-3′. The sequences were individually cloned between the BamHI and HindIII sites downstream of the U6 promoter in the pSilencer 2.1-U6 neo plasmid . To create an expression construct to overexpress F-spondin, the full-length cDNA clone of F-spondin gene in pBluescript was purchased from Open Biosystems , and the F-spondin expression construct was generated using pcDNA3.1/myc-His (-) vector (Invitrogen). Briefly, specific primers with HindIII and XhoI restriction enzyme sites were used to amplify full-length human F-spondin ORF, and the PCR product was subsequently cloned into PCRII vector by Topo TA cloning (Invitrogen). Full-length human F-spondin with Hind III and Xho I sites in the PCRII vector was then released by restriction enzymes, purified, and subcloned into the pcDNA3.1/myc-His (-) vector. The IL-1Ra plasmid was constructed by introducing the BamHI and HindIII sites at the front and back ends of the full length mouse IL-1Ra (NC_000068.7). The following primers were used to generate PCR products: 5′-GGA TCC ATG GAA ATC TGC TGG GGA CC -3′ (forward primer with BamHI site) and 5′-AAG CTT TTG GTC TTC CTG GAA GTA GA -3′ (reverse primer with HindIII site). The sequence of the PCR product was verified by a core lab at the Institute of Biomedical Sciences, Academia Sinica, and subsequently ligated into pGEM-T Easy vector  to generate the intermediate vector. The cDNA fragment encoding mIL-1Ra was then released from the intermediate vector using restriction enzymes BamHI and HindIII, and ligated into pcDNA3.1/myc-His (-) vector.The stable cell lines used in this study were established by transfection of RAW264.7 cells with F-spondin shRNA, IL-1Ra plasmid or their controls using Lipofectamine 2000 (Invitrogen). The transfected cells were further selected by G-418 sulfate  and ultimately maintained in DMEM containing 1 mg/ml Geneticin. For transient transfections, siRNAs or plasmids were transfected into cells by electroporation using Amaxa cell line nucleofector kit V (for RAW264.7 cells) or Mouse Macrophage Nucleofector Kit (for mouse BMMs) according to the manufacturer’s instructions.Total proteins were extracted from cells using protein extraction reagents containing protease inhibitors (Santa Cruz), and the concentration of protein extract was determined by Bio-Rad protein assay . Equivalent amounts of protein from each sample were resolved by SDS-PAGE and subsequently transferred onto a PVDF membrane (Millipore). The blots were blocked with TBS containing Tween 20 and 5% skim milk for 1 h and then incubated with specific primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity was detected using chemiluminescent substrate (Millipore).For cell cycle analysis, an equivalent number of wild type, vector stably transfected and IL-1Ra plasmid stably transfected RAW264.7 cells were plated into dishes and cultured for 48 h. Cells were harvested and washed with PBS once before being fixed in cold 70% ethanol for 1 h. Cell pellets were then collected by centrifugation, washed with PBS twice and treated with RNase A solution (Invitrogen). PI solution (Sigma) was added directly to cells in RNase A solution and the samples were analyzed by a FACSCalibur system  after incubation for 30 min at room temperature. The data were analyzed and plotted using ModFit LT software .2. Serial phase-contrast images were obtained every 15 min for 22 h, and the cell trajectories and migration velocities were determined using the tracking point tool of Metamorph software . The average of cell migration velocities was defined as the average of 20 subsequent cell centroid displacements/one time interval between two images.Time-lapse microscopy was performed as described previously . BrieflyP values less than 0.05 were considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001.Statistically significant difference between two groups of data was determined using a 2-tailed Student's t test. All data analyses were performed with GraphPad Prism 5.0 for Microsoft Windows . To determine the effect of CpG ODNs on IL-1Ra expression in macrophages, we first compared the response of mouse macrophage cell line RAW264.7 to CpG-A or CpG-B ODNs. The mRNA level of IL-1Ra in cells treated with CpG-B ODNs, including ODN1668, ODN2006, and ISODN6, was increased compared with that in cells treated with GpC ODN (negative control) . Cells tMock-shRNA) and a stable RAW264.7 cell line transfected with F-spondin shRNA (RAWspondin-shRNA) were generated, and the silencing effect of F-spondin shRNA was verified by RT-PCR (WT) and RAWMock-shRNA cells, but not in RAWspondin-shRNA cells (We previously found that F-spondin plays a role in regulating cytokine production . To examNA cells  and secrNA cells  of IL-1RIL-1Ra) and compared their proliferation with that of ODN1668-treated cells. Our results revealed that like ODN1668-treated cells, RAWIL-1Ra showed a proliferation rate 30% higher than ODN1668-untreated naive cells , and RAWIL-1Ra cells were subjected to flow cytometry analysis. Over-expression of IL-1Ra decreased the G0/G1 phase and increased the S phase of the cell population Click here for additional data file.S2 FigRAW264.7 cells were treated 1α M CpG ODN1668 or GpC ODN1668 for 24h, and the production of IL-6, IL-10, and NO was analyzed by ELISA . Gene expressions of TNFα and arginase 1 were analyzed by qPCR .(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Coordination between actin cytoskeleton assembly and localized polarization of intracellular trafficking routes is crucial for cancer cell migration. ARF6 has been implicated in the endocytic recycling of surface receptors and membrane components and in actin cytoskeleton remodeling. Here we show that overexpression of an ARF6 fast-cycling mutant in MDA-MB-231 breast cancer-derived cells to mimick ARF6 hyperactivation observed in invasive breast tumors induced a striking rearrangement of the actin cytoskeleton at the ventral cell surface. This phenotype consisted in the formation of dynamic actin-based podosome rosette-like structures expanding outward as wave positive for F-actin and actin cytoskeleton regulatory components including cortactin, Arp2/3 and SCAR/WAVE complexes and upstream Rac1 regulator. Ventral rosette-like structures were similarly induced in MDA-MB-231 cells in response to epidermal growth factor (EGF) stimulation and to Rac1 hyperactivation. In addition, interference with ARF6 expression attenuated activation and plasma membrane targeting of Rac1 in response to EGF treatment. Our data suggest a role for ARF6 in linking EGF-receptor signaling to Rac1 recruitment and activation at the plasma membrane to promote breast cancer cell directed migration. CancD tissue mponents –7. In admponents , 9.The protrusive and migratory potential of invasive tumor cells is enhanced by up-regulation of several genes involved in cell motility including the gene coding for the F-actin binding protein cortactin , 11. In The small GTP-binding protein ADP-ribosylation factor 6 (ARF6) is known to coordinate endocytosis, post-endocytic recycling and actin cytoskeletal organization at the plasma membrane , 17. By In this study we used a gain of function approach to mimic up-regulation of ARF6 activity reported in breast tumor cells and to unravel the mechanism through which ARF6 controls cancer cell migration. In MDA-MB-231 cells, expression of a hyperactive mutant of ARF6 (ARF6T157N) , 34 indu2.Human breast adenocarcinoma MDA-MB-231 cells (American Type Culture Collection HTB-26) were maintained in L-15 culture medium  with 2 mM glutamine (GIBCO) and 15% FBS (GIBCO) at 37°C in 1% COhttp://rsb.info.nih.gov/ij/).Cells were lysed and proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and detected by immunoblotting analysis with indicated antibodies. Bound antibodies were detected with ECL Western Blotting Detection Reagents . Densitometric quantification of bands was performed with Gels tool of ImageJ software (Antibodies used in this study are listed in Full-length rat cortactin cDNA subcloned in pDsRed1-N1 or pEGFP-N1 (Clonetech) were provided by Dr M.A. McNiven . cDNA encoding ARF6T157N  was inserted in a pDEST WPI lentiviral expression vector  through Gateway Cloning technology (Invitrogen). C-terminally GFP-tagged ARF6T157N construct was obtained by subcloning ARF6T157N cDNA into a pEGFP-N1 vector (Clontech). N-terminally-GFP tagged Rac1G12V was a gift from P. Fort . For transient expression, MDA-MB-231 cells were transfected with plasmid constructs (1 μg) by using Lipofectamine LTX (Invitrogen) or Nucleofector (Lonza) according to the manufacturer’s instructions. Cells were analyzed 48 h after transfection.pGEX constructs encoding GST or the residues 371–507 (LZII domain) of human JIP3 fused with an amino-terminal GST moiety  were purMDA-MB-231 cells or MDA-MB-231 cells stably overexpressing ARF6 wild-type, ARF6T27N or ARF6T157N were lysed in 50 mM Tris pH 7.4, 137 mM NaCl, 10 mM MgCl2, 10% glycerol, 1% Triton-X100 and a cocktail of protease inhibitors (Roche) and centrifuged at 15,000g for 15 min at 4°C. Supernatants were incubated with GST (20 μg/reaction) or GST−LZII (37 μg/reaction) for 15 min at 4°C in the presence of 0.5% BSA. Then, glutathione-Sepharose beads were added for 1 h at 4°C. Beads were washed and bound proteins were analyzed by SDS−PAGE and immunoblotting using anti-ARF6 monoclonal antibody.siRNA transfection was performed using 50 nM siRNA with Lullaby reagent (OZ Biosciences) according to manufacturer’s instructions and analyzed 72 hrs after treatment. The siRNAs used in this study are listed in For lentivirus production, HEK293T cells were transfected using GeneJuice (Novagen) with a mix of ARF6-T157N expression vector and psPAX2 (AddGene) and pVSV-G (Clonetech) packaging vectors in OPTIMEM (Invitrogen). After 72 hrs, virus-containing supernatant was collected, filtered and used for transduction of a subconfluent monolayer of MDA-MB-231 cells.MDA-MB-231 cells stably expressing ARF6T15TN were cultured on gelatin-coated coverslips prepared as described . Cells wth pixel) was at the level of the plasma membrane and fluorescence intensity along the line was measured with the Linescan tool of Metamorph software. Intensity profiles from different cells were then averaged and normalized for the highest value (set to 100).For quantification of Rac1 recruitment at cell edge, a line of 160 pixels was drown perpendicularly to the leading edge in a way that half of the line (about 80MDA-MB-231 cells and cells stably expressing ARF6T157N were incubated for 5 h on FITC-conjugated cross-linked gelatin (Invitrogen), fixed and stained for cortactin . Cells wMDA-MB-231 cells transiently transfected with DsRed-cortactin and ARF6T15TN-GFP or GFP-Rac1G12V were plated on glass-bottom MatTek dishes coated with cross-linked gelatin and kept in a humidified atmosphere at 37°C and 1% CO2. Formation of cortactin-positive rosettes was monitored by acquiring 3D time-series images by confocal spinning-disk microscopy (1 z-stack/4 or 3 s) using a Nikon Eclipse TE2000-U microscope equipped with a 60x 1.45NA oil immersion objective, a PIFOC Objective stepper, a Yokogawa CSU22 confocal unit and a Roper HQ2 CCD camera steered by Metamorph and a temperature controller. MDA-MB-231 cells stably expressing ARF6T157N and transiently transfected with GFP-cortactin were plated on MatTek dishes layered with a drop of polymerized type I collagen mixed with AlexaFluor 549-conjugated type I collagen (BD) at a final concentration of 2.5 mg/ml. Cells were incubated for 30 min at 37°C in 1% CO2 and then imaged by confocal spinning disk microscopy (2 z-stack/min).MDA-MB-231 cells stably expressing ARF6T15TN and transiently transfected with DsRed-cortactin were plated on glass-bottom MatTek dishes coated with cross-linked gelatin for two hours before being imaged. MDA-MB-231 cells were depleted with non-targeting or ARF6 siRNAs and transfected with GFP- or DsRed-cortactin, respectively. The two cell populations (red and green) were mixed and plated on gelatin-coated MatTek dishes for two hours and imaged. Simultaneous dual color TIRFM time-lapse sequences were acquired on a Nikon TE2000 inverted microscope equipped with a ×100 TIRF objective (NA = 1.47), a TIRF arm, an image splitter  installed in front of the CCD camera and a temperature controller. GFP and DsRed were excited with 491- and 560 nm-lasers, respectively , controlled for power by an acousto-optic tunable filter. Fluorescent emissions were selected with bandpass and longpass filters (Chroma) and captured by a QuantEM EMCCD camera (Roper Scientific). The system was driven by Metamorph. Images were acquired with a 1 min interval for several hours. Kymographs were obtained with Metamorph software along a line perpendicular to protruding leading edge. Membrane protrusion speed was calculated by dividing the length of the vertical axis of the kymograph representing the distance of protrusion extension by the time.For EGF stimulation experiment, MDA-MB-231 cells were transiently transfected with DsRed-cortactin, serum-starved for 16 hours and plated on glass-bottom dishes coated with cross-linked unlabeled gelatin and images were acquired with a 5 sec interval for 1 min before adding EGF (Preprotech), added directly to the medium at 100 ng/mL final concentration.MDA-MB-231 cells were serum starved for 12–16 hours and EGF was added at 100 ng/mL final concentration for the indicated times. Cells were transferred on ice and immediately permeabilized with 0.5% Triton-X100 in 4% PFA in PBS for 90 seconds, fixed in 4% PFA in PBS and stained for cortactin. For measuring ARF6 and Rac1 activation levels, we used G-LISA-ARF6 Activation Assay kit  and G-LISA-Rac1 Activation Assay kit . MDA-MB-231 cells were treated with the indicated siRNAs for 72 hours, plated on gelatin-coated wells of a 6-well plate, serum starved and EGF-stimulated as described above. EGF stimulation was blocked on ice and cells were immediately lysed with lysis buffer and processed following the manufacturer’s instructions. Briefly the G-LISA kits contain an ARF6- or Rac1-GTP-binding protein linked to the wells of a 96 well plate. Active, GTP-bound ARF6 or Rac1 in cell lysates bind to the wells while the inactive GDP-bound form is removed during washing steps. Active bound ARF6 or Rac1 were detected with ARF6 or Rac1 specific antibodies and the signal is developed with HRP detection reagents following manufacturer’s instructions.p < 0.05 considered significant.Statistical analyses were performed using Student’s or Mann-Whitney t-test two-way ANOVA and Spearman correlation test using GraphPad Prism (GraphPad Software) as specified in each figure legend with MDA-MB-231 cells stably expressing a fast-cycling mutant of ARF6 (ARF6T157N) were generated by lentiviral transduction. This mutant is characterized by increased spontaneous GDP dissociation and GTP association , thus miMDA-MB-231 cells stably expressing ARF6T157N were plated on a 2D substrate of cross-linked gelatin  and stai2, scoring 25 cells) than that of parental cells  suggesting that activated ARF6-induced cortactin-rich rosettes contribute to cell spreading. c-Src protein Tyrosine-kinase and phosphorylated-tyrosine residues (By live-cell imaging by TIRF microscopy (TIRFM) we confirmed that cortactin-positive rosettes occurred ventrally and were highly dynamic, expanding outward as waves and then disassembling in a few minutes . When thresidues . as wellresidues . were asresidues . Dynamicresidues . Focal aresidues .A role for ARF6 in lamellipodia extension was addressed by following the dynamics of cortactin-enriched rosettes in MDA-MB-231 cells by TIRFM . In contbona fide cortactin-positive invadopodia forming at the ventral plasma membrane of MDA-MB-231 cells plated on fluorescently labeled gelatin  MDA-MB-231 cells were stably transduced with lentiviral vectors expressing the indicated ARF6 variants. Cells lysates were incubated with GST alone or GST fused with the ARF6-binding domain  of human JIP3 (aa 371–507), which interacts specifically with GTP-bound ARF6. Immunoblotting analysis of ARF6 in the bound (GTP:ARF6) and input fractions. α-tubulin was used as a loading control. (B) Densitometric quantification of ARF6 bands in panel A. Values represent GTP:ARF6 levels after normalization to total ARF6 and α-tubulin levels.(TIF)Click here for additional data file.S2 Fig(A-C) MDA-MB-231 cells stably expressing ARF6T157N were plated on cross-linked gelatin, fixed and stained for the indicated markers. Images were acquired with epifluorescence microscopy. Insets are magnification of the boxed regions. Scale bars, 10 μm and 5 μm (insets). (D) Still image of a confocal spinning-disk microscopy time-lapse sequence of MDA-MB-231 cells stably expressing ARF6T157N plated on a layer of Alexa-546-conjugated type I collagen fibers (red). Cells were transiently transfected with GFP-cortactin (green). Scale bar, 10 μm. The gallery corresponds to the boxed region in the still image and show a cortactin–positive rosette (arrows) forming in association with a collagen I fiber and propagating as a wave. Time is in min. Scale bar, 5 μm.(TIF)Click here for additional data file.S3 Fig(A-E) Immunoblotting analysis of lysates of MDA-MB-231 cells stably expressing ARF6T157N treated with indicated siRNAs for 72 hrs. Antibodies are indicated on the right. Immunoblotting analysis with anti-α tubulin and anti β1-integrin was used as loading control. Asterisk in A indicates WASH-specific band. (A'-E') Densitometric quantification of bands in panels A-E. Values represent mean ± SEM of density levels of each protein normalized for β1-integrin (A'-D') or α-tubulin (E') density values from four (A' and C') and three  independent experiments. Comparisons were made with a Student’s t-test. ***, P < 0.001, **, P < 0.01 *, P < 0.05 (compared to siNT-treated cells).(TIF)Click here for additional data file.S4 Fig(A) GTP:Rac1 levels were compared in MDA-MB-231 cells vs. cells stably expressing ARF6T157N. Values are normalized mean ± SEM from replicate samples. Comparison was made with a Student’s t-test. *, P < 0.05 (compared to MDA-MB-231 cells). (B-C) Still image (B) and gallery  of a time-lapse sequence of a MDA-MB-231 cell transiently expressing Rac1G12V-GFP and cortactin-DsRed plated on cross-linked gelatin and imaged with confocal spinning disk microscopy. Scale bar, 10 μm. The gallery corresponds to the boxed region of the still image and shows formation of cortactin-positive rosettes (red) associated with Rac1G12V-GFP (green). Time is in seconds. Scale bar, 5 μm.(TIF)Click here for additional data file.S1 TableThis table provides a list of monoclonal and polyclonal antibodies used in this study, their source and specific use.(DOCX)Click here for additional data file.S2 TableThis table provides a list of siRNAs used in this study, their sequence and source.(DOCX)Click here for additional data file.S1 VideoMDA-MB-231 cells stably expressing ARF6T157N and transiently transfected with DsRed-cortactin were plated on unlabeled cross-linked gelatin and imaged by TIRFM (Nikon TE2000 inverted). Images were acquired every minute. Scale bar 10 μm.(MOV)Click here for additional data file.S2 VideoMDA-MB-231 cells stably expressing ARF6T157N and transiently transfected with DsRed-cortactin were plated on cross-linked unlabeled gelatin and imaged by TIRFM (Nikon TE2000 inverted). Images were acquired every minute. Scale bar 10 μm.(MOV)Click here for additional data file.S3 VideoMDA-MB-231 cells stably expressing ARF6T157N and transiently transfected with cortactin-GFP were plated on a layer of Alexa Fluor 549–conjugated collagen I fibrils (red) for 30 min and imaged by confocal spinning disk microscopy . Images were acquired every 30 sec. Scale bar 10 μm. The inset is a magnification of the boxed region corresponding to the gallery in (MOV)Click here for additional data file.S4 VideoMDA-MB-231 cells transiently transfected with DsRed-cortactin (red) and ARF6T157N-GFP (green) were plated on cross-linked gelatin and imaged by confocal spinning disk microscopy . Images were acquired every 4 seconds. Scale bar 10 μm. The inset is a magnification of the boxed region and corresponds to the gallery shown in (MOV)Click here for additional data file.S5 VideoMDA-MB-231 cells treated with non-targeting (left) or ARF6 (right) siRNAs and transfected with GFP- or DsRed-cortactin, respectively, were mixed, plated on unlabeled cross-linked gelatin and imaged by dual-color TIRFM (Nikon TE2000 inverted). Images were acquired every minute. Scale bar 10 μm.(MOV)Click here for additional data file.S6 VideoMDA-MB-231 cells treated with non-targeting (left) or ARF6 (right) siRNAs and transfected with GFP- or DsRed-cortactin, respectively, were mixed, plated on unlabeled cross-linked gelatin and imaged by dual-color TIRFM (Nikon TE2000 inverted). Images were acquired every minute. Scale bar 10 μm.(MOV)Click here for additional data file.S7 VideoSerum-starved MDA-MB-231 cells transiently transfected with DsRed-cortactin were plated on cross-linked gelatin and imaged by TIRFM (Nikon TE2000 inverted). Images were acquired every 5 sec. EGF was added directly in the medium after 1 min of imaging. Scale bar 10 μm.(MOV)Click here for additional data file.S8 VideoMDA-MB-231 cells transiently transfected with DsRed-cortactin (red) and Rac1G12V-GFP (green) were plated on cross-linked unlabeled gelatin and imaged by confocal spinning disk microscopy . Images were acquired every 3 seconds. Scale bar 10 μm. The inset is a magnification of the boxed region and corresponds to the gallery shown in (MOV)Click here for additional data file.


Oryza sativa, the cyclophilin 19-4 (OsCYP19-4.1) transcript was significantly upregulated in response to cold stress, and that transgenic plants were cold tolerant. Here we show that, under cold stress, OsCYP19-4 produces eight transcript variants by intron retention and exon skipping, resulting in production of four distinct protein isoforms. The OsCYP19-4 AS isoforms exhibited different cellular localizations in the epidermal cells: in contrast to OsCYP19-4.1, the OsCYP19-4.2 and OsCYP19-4.3 proteins were primarily targeted to guard and subsidiary cells, whereas OsCYP19-4.5, which consists largely of an endoplasmic reticulum (ER) targeting signal, was co-localized with the RFP-BiP marker in the ER. In OsCYP19-4.2, the key residues of the PPIase domain are altered; consistent with this, recombinant OsCYP19-4.2 had significantly lower PPIase activity than OsCYP19-4.1 in vitro. Specific protein-protein interactions between OsCYP19-4.2/3 and AtRCN1 were verified in yeast two-hybrid (Y2H) and bimolecular fluoresence complementation (BiFC assays), although the OsCYP19-4 isoforms could not bind each other. Based on these results, we propose that two OsCYP19-4 AS isoforms, OsCYP19-4.2 and OsCYP19-4.3, play roles linking auxin transport and cold stress via interactions with RCN1.Alternative splicing (AS) is an important molecular mechanism by which single genes can generate multiple mRNA isoforms. We reported previously that, in  Arabidopsis thaliana [Alternative splicing (AS) is a fundamental process of gene regulation and depends on differential splice site selection, which is regulated by exonic and intronic sequences called splicing enhancers or suppressor elements that yield multiple mRNA transcripts from the same pre-mRNA . The splthaliana  and ricethaliana , respectthaliana .Arabidopsis transcripts are regulated during acclimation to low temperature [Arabidopsis showed that the majority of cold-regulated AS events result in PTC-containing transcripts, and revealed new types of cold-responsive genes [Cold stress has a major impact on plant growth and development, significantly constraining the geographical distribution of plant species as well as agricultural quality and productivity . For adaperature ,12. Coldperature . In respperature , circadiperature , and ligperature . A recenve genes .Oryza sativa, we showed that cyclophilin 19-4 (OsCYP19-4) is involved in cold acclimation and characterized this gene in the context of developing cold tolerant crops [OsCYP19-4 transcript generated eight alternative splice forms via two types of regulated splicing events, intron retention and exon skipping. We examined the subcellular localization of these OsCYP19-4 isoforms in plant cells using GFP-fusion constructs. In addition, using yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays, we detected specific protein-protein interactions between the OsCYP19-4.2/3 AS isoforms and a regulatory α-subunit of PP2A (AtRCN1). Despite their lack of PPIase activity, these two isoforms were specifically localized to guard or subsidiary cells. Taken together, our data indicate that OsCYP19-4 is multiply alternatively spliced and that most of the splice variants encode immunophilins with nonfunctional PPIase domains. Via their interactions with RCN1, AS isoforms play regulatory roles that link auxin transport to cold stress. These findings provide insights into the functions of spliced isoforms and the fine-tuning of gene regulation in response to cold stress.In response to low temperature, plants adopt various strategies to increase their cold tolerance. In a previous study in nt crops . Here, wOsCYP19-4 expression increased dramatically in response to cold stress. Subsequent experiments revealed that, under these conditions, the OsCYP19-4 gene produces multiple RT-PCR fragments from full-length OsCYP19-4 mRNA, indicating the production of multiple isoforms by AS. OsCYP19-4 expression increased gradually during cold stress, and multiple AS variants were concurrently observed until 48 h after cold treatment . AS resulted in significant changes over a wide range of sequences, including skipping of exon 6 (OsCYP19-4.2) or retention of introns 1, 3, 4, 5 and 6 (OsCYP19-4.3-8). Retention of intron 1 or 4 introduces a PTC in OsCYP19-4.3-8, resulting in either production of truncated proteins or NMD (OsCYP19-4 intron whose splicing was not affected following exposure to cold stress. The predicted structures of OsCYP19-4 AS transcripts and AS types are represented in To confirm that these PCR products arose from s or NMD . These POsCYP19-4 genomic DNA sequence using the Aletrnative Splice Site Predictor (ASSP) [OsCYP19-4 gene have the same core sequences without any change in the sequences   and Splir (ASSP)  programsequences B. This rOsCYP19-4 generates eight AS isoforms, of which six contain PTCs. These splice variants are predicted to encode proteins with altered structural organization. A ClustalW alignment of the amino acid sequences of the proteins encoded by OsCYP19-4 AS isoforms is displayed in OsCYP19-4 produces three alternative protein isoforms resulting from intron retention and exon skipping. Among the AS isoforms, only OsCYP19-4.2 encompasses the PPIase domain. Skipping of exon 6 in the OsCYP19-4.2 transcript yields a protein 26 aa shorter than OsCYP19-4 and 6 of the 14 residues crucial for PPIase activity are altered, suggesting that the PPIase activities of OsCYP19-4.1 and OsCYP19-4.2 would be different , but not with AtGNOM . The difN.benthamiana leaves by agroinfiltration. Tobacco cells co-expressing all three complexes  displayed complementation signals in both stomata guard cells and the subsidiary cells, as in our previous BiFC experiment [Next, we performed BiFC assays to determine whether OsCYP19-4 isoforms actually interact with AtRCN1 in plant cells. To this end, split YFP fusions of OsCYP19-4 isoforms and AtRCN1 were transiently expressed in periment   were surface-sterilized with 70% alcohol for 5 min, treated with 50% clorox for 40 min, and washed eight times in sterilized water. These seeds were placed onto 1/2 MS media containing 0.4% (w/v) Phytagel and grown in a plant growth chamber under controlled conditions (12 h light/12 h dark at 28 °C and 75% relative humidity). To analyze the expression of OsCYP19-4 genes, 10-day-old rice seedlings were exposed to cold by incubation in a 4 °C room. Whole seedlings were sampled after 0, 1, 3, 6, 9, 12, 24 and 48 h of cold stress, immediately frozen in liquid nitrogen, and stored at −72 °C until RNA extraction.Seeds of rice . Purified PCR products were ligated with T-blunt vector at 16 °C overnight, and the resultant plasmids were used to transform Escherichia coli DH5α. T-blunt vector clones of OsCYP19-4 AS variants were sequenced using the M13-F and M13-R primers.Total RNA was isolated from wild-type rice seedlings at different time points using RNAiso Plus . To remove genomic DNA, total RNA was treated with RNase-free DNase Ι . RT Premix  was used for cDNA synthesis. cDNA was subjected to semi-quantitative RT-PCR, and the density of each band was normalized against http://spliceport.cbcb.umd.edu/), ASSP (http://wangcomputing.com/assp/), and NetGene2 (http://www.cbs.dtu.dk./services/netGene2/). For calling of splice sites, the sensitivity value was set at a score threshold of ≥0.2. To identify motifs in a group of intron sequences, we used the MEME tool (http://meme.ncbr.net) [http://weblogo.berkeley.edu/logo.cgi) [Prediction of the 5′ (donor site) and 3′ (acceptor site) splice sites was performed using SplicePort, an Interactive Splice Site Analysis Tool (cbr.net) , which rogo.cgi) . A logo Agrobacterium strain GV3101 and co-injected into Nicotiana benthamiana leaves. The infiltrated leaves were monitored for GFP fluorescence in epidermal cells 2 days after agro-infiltration. Fluorescence imaging was conducted using a confocal laser scanning microscope system . RFP-BiP was co-expressed with OsCYP19-4.5-GFP in N. benthamiana as an ER marker.Subcellular localization assays were performed using the binary expression vector pCAMBIA1302 containing the gene encoding green fluorescent protein (GFP). To express GFP fusions of OsCYP19-4 AS isoforms cloned into the pCAMBIA1302 vector, the respective recombinant vectors and the P19 silencing suppressor plasmid were co-transformed into The full-length CDS of OsCYP19-4 (OsCYP19-4.1) or AS forms  was cloned into vector pGBKT7. Full-length AtRCN1, N-terminal residues 1‒250 of AtGNOM (AtGNOM-N), full-length AtGNOM (AtGNOM-F), and N-terminal residues 1‒250 of OsGNOM (OsGNOM-N) were cloned into pGADT7. Primers used for cloning are described in AtRCN1 cDNA was cloned into pSPYCE-35S. The BiFC constructs and P19 silencing suppressor plasmid were co-expressed in 4-week-old Nicotiana benthamiana leaves by Agrobacterium tumefaciens infection. The leaves were monitored for YFP fluorescence 48 h post-infiltration. Tobacco leaf epidermal cells were imaged using a Zeiss R510 confocal laser scanning microscope.Interactions between AtRCN1 and OsCYP19-4 AS isoforms were investigated using the BiFC assay as previously described . OsCYP19Agrobacterium-infiltrated N. benthamiana leaves using a crude protein extraction buffer . The total protein extracts were separated on 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes . The membranes were probed with anti-HA , and anti-MYC  antibodies overnight at 4 °C. After HRP-conjugated secondary antibody probing, HRP signals were detected using a West Pico Chemiluminescent substrate  and an MyECL Imager .Total proteins were extracted from transformed yeast cells or E. coli. Protein expression was induced by the addition of IPTG to the culture, and recombinant proteins were purified using an ExiProgen™ protein synthesizer  as described in Dae Hwa Yoon et al. [Recombinant His-tagged OsCYP19-4.1 and OsCYP19-4.2 were produced using the pET28b expression vector in n et al. . Comparan et al. .


Staphylococcus epidermidis causes a large number of catheter-related sepsis infections annually in the United States. We present the 2.54-Mbp complete genome assembly of reference strain S. epidermidis AmMS 205, including a single 37.7-kbp plasmid. The annotated assembly is available in GenBank under accession numbers CP009046 and CP009047. Staphylococcus epidermidis has been called an “accidental pathogen,” because it evolved to be a commensal rather than pathogenic microbe . microbe . Unlike response . Despiteannually . In thisHigh-quality genomic DNA was extracted from a purified isolate using Qiagen Genome Tip-500 at USAMRIID-DSD. Specifically, a 100-mL bacterial culture was grown to the stationary phase and nucleic acid was extracted as per the manufacturer’s recommendations. Sequence data included both 100-bp Illumina standard data (307-fold genome coverage) and long-insert 454 paired-end data  . We then7–S. epidermidis AmMS 205 includes a 2,500,626-bp circular chromosome (32.1% G+C content) and a 37,688-bp circular plasmid (27.3% G+C content) and was annotated using an Ergatis-based workflow (The complete genome assembly of workflow  at LANL CP009046) and plasmid (CP009047) are publicly available in the NCBI database.The chromosome (


The PI-3 kinase (PI-3K)/mTOR pathway is critical for cell growth and proliferation. Strategies of antagonising this signaling have proven to be detrimental to cell survival. This observation, coupled with the fact many tumours show enhanced growth signaling, has caused dual inhibitors of PI-3K and mTOR to be implicated in cancer treatment, and have thus been studied across various tumour models. Since PI-3K (class-I)/mTOR pathway negatively regulates autophagy, dual inhibitors of PI-3K/mTOR are currently believed to be autophagy activators. However, our present data show that the dual PI-3K/mTOR inhibition (DKI) potently suppresses autophagic flux. We further confirm that inhibition of Vps34/PI3KC3, the class-III PI-3K, causes the blockade to autophagosome-lysosome fusion. Our data suggest that DKI induces cell death independently of apoptosis and necroptosis, whereas autophagy perturbation by DKI may contribute to cell death. Given that autophagy is critical in cellular homeostasis, our study not only clarifies the role of a dual PI-3K/mTOR inhibitor in autophagy, but also suggests that its autophagy inhibition needs to be considered if such an agent is used in cancer chemotherapy. Macroautophagy (autophagy hereafter), is a bulk degradation system that mediates clearance of unwanted cytoplasmic constituents, including aberrant long-lived cytoplasmic proteins, intracellular pathogens and damaged organelles such as mitochondria -3. AutopThe ULK1/2-Atg13-FIP200 complex senses the signals for autophagosome initiation and stimulates the downstream class-III PI-3 kinase (PI-3K), Vps34 , 6. BeclThe ligation of growth factors to their receptors activates the class-I PI-3K and AKT kinase, that in turn activate mammalian target of rapamycin (mTOR) by inhibition of mTOR negative regulator TSC2, thus stimulating cell growth and proliferation . mTOR haClinicalTrials.gov ID: NCT01495247) and renal cell carcinoma patients (NCT01453595), as well as castration-resistant forms of prostate cancer (NCT01717898) and pancreatic neuroendocrine tumours (NCT01628913). Such dual kinase inhibitors will be further exploited for cancer therapy, with new agents entering clinical trials [The PI-3K/mTOR pathway is over-activated in many cancer cells , 21. Thel trials .Dual inhibitors of PI-3K/mTOR are currently viewed as autophagy activators , 32, 33 As PI-3K/Akt/mTOR negatively regulates autophagy, DKI has been suggested to stimulate autophagy , 32, 33.Since autophagy encompasses the delivery of LC3-II-associated autophagosomes to lysosomes and their subsequent breakdown (‘autophagy flux’) , 38, incOur data unexpectedly suggested that DKI may impair autophagy. To explore this possibility further, we used additional methods of assessing autophagy flux. p62 recruits cargo to be engulfed by autophagosomes and is subsequently degraded by lysosomal enzymes after autophagosome-lysosome fusion . As p62 As the DKI drug PI-103 appeared to disrupt autophagy flux, we next sought to investigate whether this effect was the result of changes to lysosome acidity or integrity. Acridine orange (AO) is a lysosomotropic metachromatic fluorochrome that emits red fluorescence under acidic pH, which is normally in late endosomes/lysosomes, and green fluorescence otherwise . Thus, rOur data indicated that PI-103 inhibits autophagy flux. To assess the consistency of this DKI-induced defect on autophagic flux, we further examined another prominent PI-3K/mTOR dual inhibitor, NVP-BEZ-235 (BEZ-235). Treatment with BEZ-235 increased LC3-II levels Figure . SimilarTo further test whether DKI impairs autophagosome-lysosome fusion and subsequently causes autophagosome accumulation, we again utilized mRFP-GFP-LC3 stably expressing cells for BEZ-235 treatment. As with PI-103, BEZ-235 treatment caused autophagosome number to largely increase, while autolysosome number dramatically decreased, indicating autophagosome-lysosome fusion was blocked Figure . The chaAs the lysosomal effects of DKI had not been previously reported, we were interested in determining the mechanism by which these effects were mediated. To dissect DKI-targeted autophagy pathways, we employed a pharmacological strategy by using the single treatments of mTOR inhibitor rapamycin (Rap), and PI-3K inhibitor LY294002 (LY), or the combinatory treatment of Rap+LY. Both Rap and LY are widely used in autophagy research, with Rap inducing autophagosome synthesis , and LY DKI treatments are known to be able to induce cell death, hence are attractive for tumour therapy , 31, 33.Previous studies, based on the fact that DKI induces production of apoptosis markers, suggested that DKI drug treatments execute cell death via the caspase-mediated apoptosis pathway , 60. HowAs apoptosis did not appear to be a major effector of PI-103 cytotoxicity, we next explored the involvement of another programmed cell death pathway, necroptosis. A key positive regulator of this process is RIP1, meaning that inhibition of this protein also prevents necroptosis onset , 64. We As neither apoptosis nor necroptosis suppression significantly alleviated DKI-induced cell death, we reasoned that it was possible one pathway could compensate for the other. However, synergistic use of zVAD and Nec had no effect on cell death Figure  or viabiIn this study, we show that two dual inhibitors of mTOR and PI-3K, PI-103 and NVP-BEZ-235, inhibit autophagy, suggesting that this effect is common across all such agents. Interestingly, our data also strongly suggest DKI-induced cell death is independent of apoptosis and necroptosis, and inhibition of autophagic flux by DKI may contribute to its-induced cell death.DKI appears to inhibit autophagy by blocking autophagosome maturation. It is believed that inhibition of mTOR induces autophagosome formation, while inhibition of Vps34 blocks autophagosome-lysosome fusion, resulting in decreased autophagic flux, and accumulations of non-degraded autophagosomes. This finding differs from previous observations using PI-103, which suggest the drug increases autophagy activity . While tAlthough Vps34 is critical for generating PI(3)P in the process of autophagosome formation , our datPreviously DKI-induced cell death was suggested to be apoptosis-dependent , 60. HowThe PI-3K/mTOR dual inhibitors hold a promise for treating cancers , 25, 28.Rabbit polyclonal antibodies: anti-LC3  ; anti-phospho-70S6K (Thr389)  ; anti-Akt (pan)  ; anti-phospho-Akt (Ser473)  ; anti-ULK1 (pan)  ; anti-phospho-ULK1 (Ser757)  ; anti-phospho-4E-BP1 (Thr37/46)  ; anti-Atg12 (1:500) ; anti-RIP1  ; anti-caspase 3  ; anti-Cleaved caspase 3  ; anti-caspase 8  ; anti-caspase 9  ; anti-LAMP1  ; anti-mTOR 7C10 (1:500) ; anti-PI3 Kinase Class III (Vps34) (1:500) ; anti-actin  . Goat polyclonal antibody: anti-cathepsin D (1:200) . Anti-mouse monoclonal antibodies: anti-GAPDH  ; anti-p62  ; anti-tubulin  . Bafilomycin A1 (Baf) (19-148) was purchased from Millipore. Chloroquine (CQ) (C6628), 3-methyladenine (3-MA) (M9281), rapamycin (Rap) (R0395), staurosporine (STS) (S5921), cycloheximide (C4859), artesunate (ART) (A3731) and necrostatin-1 (Nec) (N9037) were purchased from Sigma. zVAD-fmk (zVAD) (627610) was a product of Merck. PI-103 (528100) was from EMD4Biosciences, and NVP-BEZ-235 (10565) was purchased from Cayman Chemical. LY294002 (LY) (9901) was purchased from Cell Signaling Technology, and Human TNF-α was from Invitrogen . siRNAs were from Invitrogen (Ambion) or Dharmacon.Bax/Bak WT and double-knockout (DKO) MEFs, used in our previous study , RT4-D6PCells were split to 60-80% confluence and incubated in antibiotic-free DMEM containing 10% FBS. siRNAs were transfected with Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. siRNAs were used at a final concentration of 50nM. Non-targeting siRNA was the control siRNA. Cells were maintained in 10% FBS DMEM media without antibiotics for 24 hours post transfection, and then incubated in DMEM with 10% FBS for further 24-48 hours after medium change. Human RIP1 siRNA (#J-004445-07) and caspase-8 siRNA (#J-003466-16) were obtained from Dharmacon. Human siRNA sequences: FRAP1 (mTOR): (Sense: 5′-GGGCAUGAAUCGGGAUGAU-3′) (Invitrogen); PIK3C3 (Vps34): (Sense: 5′-GGGAAGAGAGAACAAAAGA-3′) (Invitrogen); caspase-3: (Sense: 5′-UGGAUUAUCCUGAGAUGGG-3′) (Invitrogen); caspase-9: (5′-Sense: GGUUCUCAGACCGGAAACA-3′) (Invitrogen).1. ATP assays. Cell survival was determined with Cell Titer-Glo Luminescent Cell Viability Assay kit  to measure ATP levels according to the manufacturer's instruction. Briefly, 100ul of Cell Titer-Glo reagent was added to the culture medium. Cells were placed on a shaker for 5 min and then incubated at room temperature for 10 min. The SPECTRA Max M5 reader was used for Luminescent reading.2. MTT assays. MTT was purchased from Invitrogen. Briefly, 10ul of a 12mM MTT stock solution was added to culture medium and incubated at 37°C for 4 hours. Medium was exchanged with 100ul DMSO, mixed by pipetting, and placed on a plate shaker for 10 min. Absorbance was read at 562nm and a reference measurement at 650nm using the TECAN GENios V4.62-07/01 microplate reader  and XFLUOR4 Version V 4.51 software (Tecan).Cell proliferation was measured using BrdU Cell Proliferation Assay kit  and performed following the manufacturer's instructions. Absorbance was read at 450nm using the TECAN GENios V4.62-07/01 microplate reader  and XFLUOR4 Version V 4.51 software (Tecan).2) at 1×106 cells/ml. We transferred 100ul of cells to a FACS tube, added 5ul propidium iodide (Sigma) and incubated for 15 min at room temperature. We added 200ul of 1xbinding buffer into each tube and analysed by flow cytometry.Cells were trypsinised and washed twice with cold PBS, then resuspended in 1x binding buffer  for 30 min at room temperature, and incubated with primary antibodies overnight at 4°C. Cells were subjected to three 10 min PBS washes, incubated with secondary antibodies for 30 min, then followed by an additional three 10 min PBS washes. Slides were mounted with DAPI (3ug/ml).HeLa cells stably expressing mRFP-GFP-LC3 were treated with compounds at the indicated concentrations. After 24 hours, cells were fixed in 2% PFA for 5 min. Cellomics (Arrayscan VTI) was used to score green and red vesicles. Green vesicles are considered to be autophagosomes and red vesicles are considered to be both autophagosomes and autolysosomes. The number of autolysosomes was achieved by subtracting the number of green vesicles from that of the red vesicles.In experiments requiring a precise assessment of vesicle number, the number of vesicles per cell in GFP or RFP-positive cells was determined. Approximately 100 cells per sample were counted for triplicate samples, as described previously  or otherFollowing drug treatment incubation, cells were stained with 2.5μg/ml acridine orange (Fisher) for 15 min at 37°C and washed twice with PBS. Live cell imaging was performed on a Leica TCSSP8 with a 1.4nm 63x objective. The argon laser at 488nm was used for excitation with emission collected simultaneously at both 505-570nm and 615-754nm.Following drug treatment, cells were stained with 1uM LysoSensor Green DND-189 (Fisher) for 30 minutes at 37°C. The media was exchanged and then live cell imaging was performed by confocal microscopy using an excitation wavelength of 488nm.2, 0.1mM EGTA, 0.5mM DTT, 0.5mM PMSF and 10ug/ml proteinase inhibitor). This mix was passed through a 21-gauge needle 10 times and kept on ice for 45 min. After removing some of the lysate , samples were centrifuged . The supernatant (cytosolic fraction) was collected. The pellet (membrane fraction) was washed twice with fractionation buffer  before running with SDS-PAGE.Cell fractionation was performed following Jahreiss et al . BrieflyRNA isolation was performed with TRIzol reagent as instructed (Invitrogen). For qPCR analysis, 1ug RNA was reverse transcribed  using cycles of 25°C (10 min), 37°C (120 min) and 85°C (5 min). cDNA templates were then used in qPCR with LightCycler 480 DNA SYBR Green I Master kit (Roche) in LightCycler 480 II system (Roche). All primers were from Sigma and used at 0.5uM. Actin was used as a control to normalize the data. Actin primers: 5′-ACTGGCATCGTGATGGACTC-3′ (forward) and 5′-TCAGGCAGCTCGTAGCTCTT-3′ (reverse); LC3-B primers: 5′-ACGATACAAGGGTGAGAAGCA-3′ (forward) and 5′-GTCCGTTCACCAACAGGAAG-3′ (reverse); p62 primers: 5′-AGATGAGGAAGATCGCCTTG-3′ (forward) and 5′-GGCATCTGTAGGGACTGGAG-3′ (reverse); mTOR primers: 5′-TAAGAAAACGGGGACCACAG-3′ (forward) and 5′-TGAGAGAAGTCCCGACCAGT-3′ (reverse); Vps34 primers: 5′-TCCTTGATGGTTGATGCAAA-3′ (forward) and 5′-CAGCAAAAAGAGCATGGACA-3′ (reverse).To quantify protein band density, the relevant specified bands were analysed using PhosphoImage software. The relative value was computed.P < 0.001, **, P < 0.01, *, P < 0.05, NS, not significant).T-test was used and P-values were determined by unconditional logistical regression analysis by using the general loglinear option of SPSS 9.1 software  (***,


JUN and p53 pathways and inhibition of genes linked to the TNF pathway. Early cellular effects activated by SB225002 included the up-regulation of GLIPR1, a p53-target gene shown to have pro-apoptotic activities in prostate and bladder cancer. Silencing of GLIPR1 in B- and T-ALL cell lines resulted in increased resistance to SB225002. Although SB225002 promoted ROS increase in ALL cells, antioxidant N-Acetyl Cysteine pre-treatment only modestly attenuated cell death, implying that the pro-apoptotic effects of SB225002 are not exclusively mediated by ROS. Moreover, GLIPR1 silencing resulted in increased ROS levels both in untreated and SB225002-treated cells. In conclusion, SB225002 induces cell cycle arrest and apoptosis in different B- and T-ALL cell lines. Inhibition of tubulin function with concurrent activation of the p53 pathway, in particular, its downstream target GLIPR1, seems to underlie the anti-leukemic effect of SB225002.Acute Lymphoblastic Leukemia (ALL) is the most frequent childhood malignancy. In the effort to find new anti-leukemic agents, we evaluated the small drug SB225002 (N-(2-hydroxy-4-nitrophenyl)-N’-(2-bromophenyl)urea). Although initially described as a selective antagonist of CXCR2, later studies have identified other cellular targets for SB225002, with potential medicinal use in cancer. We found that SB225002 has a significant pro-apoptotic effect against both B- and T-ALL cell lines. Cell cycle analysis demonstrated that treatment with SB225002 induces G2-M cell cycle arrest. Transcriptional profiling revealed that SB225002-mediated apoptosis triggered a transcriptional program typical of tubulin binding agents. Network analysis revealed the activation of genes linked to the  Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood. We previously reported that the leukemic bone marrow (BM) microenvironment has increased levels of chemokine (C-C motif) ligand 2 (CCL2) and interleukin-8 (IL-8), and that these chemokines have a positive impact on BM mesenchymal stem cells, but no direct effect on ALL cells . In our in vivo  or SB225002 with the following concentrations: REH and RS4;11 [10 μM]; Jurkat and TALL-1 [3.125 μM]. After 24 h of treatment, cells were centrifuged and fixed in cold 70% ethanol, washed twice with PBS and stained with 1 ml of propidium iodide solution  supplemented with 50 μl RNase A (50 mg/ml) for 1 h at room temperature and analyzed with a FACSCanto cell cytometer . At least 20,000 cells were collected and the cell cycle profiles were calculated using the BDFACSDiva (Becton Dickinson).GLIPR1 B- and T-ALL cells were analyzed at the timepoints 0 h, 24 h and 48 h. Cells were then washed once with PBS and labeled with annexin-V-FITC and propidium iodide-PE  for 15 min at room temperature. Cells were analyzed with a FACSCanto flow cytometer (Becton Dickinson) using the BDFACSDiva Software (Becton Dickinson).B-ALL  and T-ALL (Jurkat and TALL-1) cells were treated with DMSO [0.1%] or SB225002 [5 or 10 μM] for 24 h. Untreated sh.scramble and sh.GLIPR1 knockdown and scramble (negative control sh.RNA) cell lines. Treatments were performed for 24 h. For experiments using N-Acetyl Cysteine (NAC), media was buffered with HEPES 20 mmol/L, cells were pre-incubated with NAC [10 mM] for 3h and then treated with SB225002 [5 or 10 μM] for 6 or 24 h  and 48 h . Cells were washed twice with PBS and stained with 20 μM of 2',7'-dichlorodihydrofluorescein diacetate  for 30 min at 37°C. Analysis was performed after the acquisition of 50,000 events on a BD FACSCanto flow cytometer (Becton Dickinson).B-ALL , T-ALL (Jurkat and TALL-1) and PHA-stimulated normal lymphocytes were treated with DMSO [0.1%] or SB225002 [5 μM and 10 μM]. Similar experiments were also carried out with the per well. Viable cells were counted on BD FACSCanto flow cytometer at different time points. Cell viability assays were performed in 96-well micro-titer plates containing 30,000 cells per well for the ALL cells lines and 200,000 cells per well for the PHA-stimulated normal lymphocytes using the MTT reagent (Sigma-Aldrich). The formazan dye formed by the viable cells was quantified by measuring the absorbance of the dye solution at 590 nM.Proliferation assay was performed in 96-well micro-titer plates containing 10,000 ALL cells GLIPR1 gene were obtained from Sigma-Aldrich (SHCLNV-NM_006851). Clones TRCN0000123175 and TRCN0000123176 (Sigma-Aldrich) were used for screening, and clone TRCN0000123176 chosen for subsequent experiments. As negative control, cells were transduced with the MISSION Non-Target shRNA Control Vector (Sigma-Aldrich). After 72 h, infected ALL cell lines REH, RS4;11, Jurkat and TALL-1 were selected with 1.5 μg/mL  or 2.5 μg/mL  puromycin during 10 days. Bulk cells, cultured 1 week without puromycin, were used for experiments.The lentiviruses encoding shRNA sequences against the 7 cells were injected in NOD/SCID (NOD.CB17-Prkdcscid/J) mice  by the tail vein. ALL engraftment was monitored as previously described  for 24 h. Most of the untreated ALL cells were non-apoptotic (Annexin-V negative/PI negative), while upon treatment with SB225002 a marked increase in the number of early (Annexin-V positive/PI negative) and late apoptotic (Annexin-V positive/PI positive) cells was observed in both B- and T-ALL cells .50 dose] for 6 h and 9 h. Transcriptional profiling analysis revealed 174 induced and 41 repressed genes that were commonly modulated after both 6 h and 9 h of treatment  database (build 02), which included 6,100 genome-wide expression profiles representing 1,309 compounds. As shown in JUN and p53 pathways and inhibition of TNF pathway  were also analyzed using Ingenuity Pathway Analysis. As shown in  pathway .JUN pathway is directly and indirectly involved in apoptosis induction  intraperitoneally, once a day, 5 days a week, during 4 weeks. (B) Kaplan-Meier survival curve of mice treated with vehicle or SB225002 [10 mg/Kg] as described above. P value was calculated using Log-rank test.(TIF)Click here for additional data file.S2 Fig(A) REH and (B) Jurkat cells protein levels were investigated by Western blot analysis. Cells treated with SB225002 [IC50] or DMSO  for 3h, 6 h, 9 h or 12 h, as indicated. GAPDH was used as loading control. Control = DMSO (vehicle control); SB = SB225002 treatment.(TIF)Click here for additional data file.S3 Fig(A)c-JUN and (B)BACH2 were performed by quantitative PCR in Jurkat cells. Expression values were calculated considering vehicle control (DMSO) as 100%. Control = DMSO (vehicle control); SB = SB225002 treatment.Gene expression analysis for (TIF)Click here for additional data file.S4 Fig(B) Validation of GLIPR1 knockdown in the different B-ALL  and T-ALL (Jurkat and TALL-1) cell lines performed with clone TRCN0000123176 (Sigma-Aldrich).Expression values were calculated considering transduction control as 100%. G-KD#1 corresponds to clone TRCN0000123175 and G-KD#2 to clone TRCN0000123176 (both from Sigma-Aldrich). (TIF)Click here for additional data file.S5 Fig(A) Relative proliferation of REH, RS4;11, Jurkat and TALL-1 cells upon sh.RNA knockdown of GLIPR1 (sh.GLIPR1) in comparison to control Scramble (sh.Scr). Number of viable cells was measured by the MTT assay and normalized to time-point zero. (B) Annexin-V and propidium iodide flow cytometry analyses of B-ALL  and T-ALL (Jurkat and TALL-1) scramble or GLIPR1-knockdown cells at different timepoints as indicated. S = scramble transfection control; G-KD = cells infected with GLIPR1-shRNA lentiviral particles (Sigma-Aldrich).(TIF)Click here for additional data file.S6 FigGLIPR1-shRNA lentiviral particles (Sigma-Aldrich). P values were calculated using two-tailed Student’s t-test.Cells were treated for 24 h. Percentage of viable cells was analyzed by flow cytometry (annexin-V staining negative population). S = scramble transfection control; G-KD = cells infected with (TIF)Click here for additional data file.S7 FigCells were treated for 24 h. Control = DMSO (vehicle control); SB = SB225002 treatment.(TIF)Click here for additional data file.S8 FigGLIPR1-shRNA lentiviral particles (Sigma-Aldrich). P values were calculated using two-tailed Student’s t-test.Cells were pre-incubated with NAC [10 mM] for 3 h prior to the SB225002 treatment. B-ALL  cells were treated with SB225002 [10 μM] and T-ALL (Jurkat and TALL-1) were treated with SB225002 [5 μM] for 6 h or 24 h as indicated. S = scramble transfection control; G-KD = cells infected with (TIF)Click here for additional data file.S1 TableTranscriptional profiling analysis was performed in Jurkat (T-ALL) cells.(DOCX)Click here for additional data file.