Gpr43-deficient mice and transgenic mice with adipose-tissue-specific overexpression of GPR43. We found that GPR43 activation by SCFA resulted in induction of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) in anti-inflammatory M2-type macrophages within adipose tissue. By contrast, this effect was not noted in inflammatory M1-type macrophages, suggesting that GPR43 plays distinct functions depending on macrophage types. Local TNF-α signaling derived from steady-state adipose tissue is associated with proper tissue remodeling as well as suppression of fat accumulation. Thus, GPR43-involving mechanism that we have identified supports maintenance of adipose tissue homeostasis and increase in metabolic activity. This newly identified facet of GPR43 in macrophages may have clinical implications for immune-metabolism related episodes.The regulation of inflammatory responses within adipose tissue by various types of immune cells is closely related to tissue homeostasis and progression of metabolic disorders such as obesity and type 2 diabetes. G-protein-coupled receptor 43 (GPR43), which is activated by short-chain fatty acids (SCFAs), is known to be most abundantly expressed in white adipose tissue and to modulate metabolic processes. Although GPR43 is also expressed in a wide variety of immune cells, whether and how GPR43 in adipose tissue immune cells regulates the inflammatory responses and metabolic homeostasis remains unknown. In this study, we investigated the role of GPR43 in adipose tissue macrophages by using  Metabolic disorders such as obesity and type 2 diabetes have become a global epidemic, with incidence rates of over 30% in most Western countries. The obeGpr43-/-) mice develop obesity when they are on high fat and normal chow diets, and GPR43 activation increases energy expenditure and preferentially enables fat consumption via inhibition of insulin signaling in the WAT[G-protein-coupled receptor 43 (GPR43), also called free fatty acid receptor 2 (FFAR2), binds short-chain fatty acids  produced by the microbial fermentation of carbohydrates. Emerging research has suggested that GPR43, which is expressed in adipose tissue, pancreatic islets, and gastrointestinal tract, is involved in modulating metabolic processes–20. For n the WAT. Moreoven the WAT–25.The role of GPR43 in the regulation of inflammatory responses remains obscure or even controversial. It has been reported that GPR43 signaling reduces inflammatory mediator production and affects inflammatory leukocyte migrations. HoweverGpr43-/- mice and transgenic mice with adipose-tissue specific overexpression of GPR43. We found that GPR43, which is expressed in anti-inflammatory M2-type macrophages within the WAT, critically contributed to the induction of tumor necrosis factor-α (TNF-α), depending on SCFA stimulation. However, this effect of GPR43 was not seen in the other types of macrophages such as inflammatory M1-type macrophages. Inflammatory signaling in steady-state adipose tissue has been shown to be necessary for tissue homeostasis and for enhancing insulin sensitivity[In this study, we investigated the role of GPR43 in the adipose tissue macrophages by using nsitivity. Thus, oGpr43 mRNA expression in each fraction by quantitative reverse-transcription PCR (qRT-PCR). Although high Gpr43 mRNA levels were noted in MAs as previously reported[Gpr43 mRNA expression in the SVF, albeit the expression levels were lower than those in MAs -fed mice -fed mice into mature adipocytes (MAs) and the SVF , and ana reported, we coule in MAs . Similare in MAs , and Gprfed mice . These raP2-Gpr43TG)[aP2-Gpr43TG mice were significantly lower than those of wild-type (WT) mice in the NC-fed condition. However, the weights of other tissues were not dramatically different between WT and aP2-Gpr43TG mice, indicating that aP2-Gpr43TG mice did not have ectopic fat deposition  and increased sensitivity during insulin tolerance tests (ITT)  promoter-driven adipose-specific human Gpr43 transgenic mice (-Gpr43TG). In agre-Gpr43TG), the bodts (ITT) . We haven WT mice. TherefoaP2-Gpr43TG mice. Surprisingly, mRNA expression of the pro-inflammatory cytokine Tnfα was much higher in aP2-Gpr43TG mice than WT mice, even if aP2-Gpr43TG mice were leaner (aP2-Gpr43TG mice (F4/80 (macrophage marker) and Mcp-1  did not differ between both types of adipose tissue  mRNA induction  subsets and non-macrophage (F4/80-CD11b-) subsets by using a flow cytometry approach , arginase-1 (Arg1), and CD301 and secrete anti-inflammatory cytokines including IL-10 and IL-1 receptor antagonists[Inos), TNF-α, IL-6, IL-1β, IL-12, and MCP-1[Gpr43-/- mice into M1- or M2-type macrophages and examined the inflammatory responses, depending on SCFA stimulation in vitro. We confirmed that robust Inos mRNA induction occurred in M1-polarized macrophages and that induction of Mrc1 mRNA expression was not detectable, whereas the converse was observed in M2-polarized macrophages  signaling[Gpr43-/- M2-type macrophages revealed that extracellular signal-regulated kinase 1 (Erk1) was phosphorylated in WT macrophages after acetate stimulation, but this was strongly suppressed in Gpr43-/- macrophages  stimulated with acetate or propionate. In contrast to the findings for M2-type bone marrow-derived macrophages, Tnfα mRNA expression was suppressed in WT thioglycollate-elicited PECs, depending on SCFA stimulation; this was not observed in Gpr43-/- thioglycollate-elicited PECs  than in non-treated Raw264.7 cells in vitro  in murine islets revealed that selective GPR43 signaling through either Gi/o or Gq translated into disparate effects on GSIS[The mechanism underlying the difference between M1- and M2-type macrophages with respect to different GPR43-mediated inflammatory responses is not known. GPR43 couples to either Gs on GSIS. Since Gin vitro depending on the types of immune cells. Furthermore, in aP2-Gpr43TG mice, high levels of TNF-α production were seen only in the WAT but not in the liver and muscle, indicating that the situation is more complex in vivo. For instance, recent findings suggest that (a) adipocytes collaborate with certain immune cells and directly regulate the activation and proliferation of adipose immune cells by secreting various adipokines such as adiponectin and MCP-1 [Recent studies have implicated involvement of GPR43 in chronic inflammatory diseases such as obesity, colitis, and cancer,22,43. Hnd MCP-1 ,45 and tnd MCP-1 . Thus, sIn conclusion, the findings of our previous study and our Gpr43-/- was described previously[aP2-Gpr43TG mice were generated as previously[C57BL6/J mice were housed under a 12-h light–dark cycle and given regular chow . All experimental procedures involving mice were performed according to protocols approved by the Committee on the Ethics of Animal Experiments of the Tokyo University of Agriculture and Technology. (Permit Number: 28–87). For HFD studies, 4-week-old male mice were placed on a D12492 diet  for 12 weeks. The generation of reviously; the micreviously. For antPremix Ex Taq II (TAKARA) and the ABI7300 apparatus (Applied Biosystems). Primer sequences for Gpr43 (mouse), Tnfα (mouse) and GPR43 (human) have been described previously[F4/80 (mouse), 5’- GATGTGGAGGATGGGAGATG -3’ (forward) and 5’- ACAGCAGGAAGGTGGCTATG -3’ (reverse); Mcp-1 (mouse), 5’- AATCTGAAGCTAATGCATCC -3’ (forward) and 5’- GTGTTGAATCTGGATTCACA -3’ (reverse); Il6 (mouse), 5’- GGAGTACCATAGCTACCTGG -3’ (forward) and 5’- AGGAATGTCCACAAACTGAT -3’ (reverse); Inos (mouse), 5’- TGGTGGTGACAAGCACATTT -3’ (forward) and 5’- AAGGCCAAACACAGCATACC -3’ (reverse); Mrc1 (mouse), 5’- CAAGGAAGGTTGGCATTTGT -3’ (forward) and 5’- CCTTTCAGTCCTTTGCAAGC -3’ (reverse); 18S (mouse), 5’- ACGCTGAGCCAGTCAGTGTA -3’ (forward) and 5’- CTTAGAGGGACAAGTGGCG -3’ (reverse), Pparg2 (mouse), 5’- GCTGTTATGGGTGAAACTCTGG -3’ (forward) and 5’- TTCTTGTGAAGTGCTCATAGGC -3’ (reverse), Cd45 (mouse), 5’- tcgtgcccaaacaaattaca -3’ (forward) and 5’- taggcttaggcgtttctgga -3’ (reverse).Total RNA was extracted using an RNeasy Mini Kit (Qiagen) and subjected to DNase treatment (Invitrogen), and reverse transcription were performed using Moloney murine leukemia virus reverse transcriptase (Invitrogen). Quantitative reverse transcriptase PCR (qRT-PCR) analysis was performed with using SYBR reviously. Other pMurine TNF-α was measured by ELISA. TNF-α ELISA kits was obtained from R&D systems.Epididymal, mesenteric and subcutaneous adipose depots were dissected from mice and minced in sterile PBS. The tissues were then digested with 1 mg/ml collagenase type I (Sigma) in PBS at 37°C for 30 min with gentle shaking. The suspension was then passed through a 100-μm mesh to remove undigested clumps and debris. The flow-through was allowed to centrifuge at 300rpm for 3 min to separate the floating MAs fraction. After removing MAs fraction, the SVF was collected by centrifugation at 1500rpm for 10 min. The resultant isolated cells were subjected to FACS analysis.+CD11b+F4/80+ cells) and non-macrophages immune cells (CD45+CD11b-F4/80- cells) were each sorted by a MoFo XDP (BECMAN COULTER).The pellet containing the SVF was washed once with ice-cold FACS buffer (2% FCS in PBS). After washing, the cells were stained with phycoerythrin (PE)-Cy7 conjugated anti-CD45 (BioLegend), allophycocyanin (APC) conjugated anti-CD11b (BioLegend) and biotin conjugated anti-F4/80 (BioLegend) following by incubation with Alexa Fluor 488 conjugated streptavidin (Invitrogen) as secondary antibody. Macrophage subsets  per gram of body weight (1.5 mg / g) intraperitoneally. For ITT, 3-h-fasted mice were given human insulin  intraperitoneally. Blood glucose concentration was monitored using a OneTouch Ultra before injection and at 15, 30, 60, 90 and 120 min after injection.M2 polarized bone marrow derived macrophages were cultured in serum-free RPMI 1640 (Gibco) for 3 h, and then stimulated with acetate . After 10 min, cells were lysed in TNE buffer and centrifuged at 14,000 g for 20 min at 4°C. The supernatants were resolved by SDS gel electrophoresis and blotted onto a nitrocellulose membrane. Primary antibodies used were follows: ERK1/2  and phosphorylated ERK1/2 . The secondary antibody used was a horseradish peroxidase-conjugated goat anti-rabbit antibody . Immunoreactive bands were visualized using an enhanced chemiluminescence detection system. Image J  was used to quantify the integrated density of each band.Adipose tissue was fixed in 10% Formalin Neutral Buffer Solution (Wako). Fixed sections were embedded in paraffin, sectioned and stained with the following primary antibodies: F4/80  and Caveolin1 . The secondary antibody used was a Alexa Fluor 488-conjugated anti-rat antibody  or Alexa Fluor 555-conjugated anti-mouse antibody , respectively. At the end of the staining, slides were washed and incubated with DAPI (Sigma) and mounted with Fluoromount-G (Southern Biotech). Fluorescence images were obtained with a fluorescence microscopy LSM710 (Carl Zeiss).The bone marrow cells from mice were isolated and incubated in RPMI 1640 (Gibco) supplemented with mouse macrophage colony-stimulating factor mouse  and 10% fetal bovine serum (FBS). The medium was replaced with fresh RPMI 1640 medium containing with MCSF (50 ng/mL) and 10% FBS every two days. On day 6 in culture, cells were activated (M1 condition) with MCSF (50 ng/mL), LPS  and IFN-γ , alternatively activated (M2 condition) with MCSF (50 ng/mL) and IL-4  for 24 h. After polarization, cells were stimulated with acetate (10 mM) for 7 h.Mice were euthanized 72 h after intraperitoneally injection of 3 ml 4% thioglycollate. Peritoneal cells were harvested by injecting 7 mL of FACS buffer (2% FCS in PBS) into PerC. Macrophages were enriched by adherence by plating peritoneal cells in RPMI 1640 medium for 3 h. Non-adherent cells were removed, then treated with serum-free for 12 h later and stimulated with acetate (10 mM) for 7 h.Mouse macrophage cell line (Raw264.7 cells) were cultured with Dulbecco’s modified Eagle’s medium  containing 1% penicillin–streptomycin solution (Gibco) and 10% FBS. Raw cells were stimulated with LPS  for 24 h and then harvested to isolate RNA.18. Then, collected and pooled ether layers were transferred to glass vials for GC-MS analysis. Analyses were performed with a GCMS-QP2010 Ultra (Shimadzu) as described previously18.Sample preparation protocol for SCFA determination in serum modified the method described previouslyValues are presented as the mean ± s.e.m. Differences between groups were examined for statistical significance using Student’s t-test (two groups) or one-way analysis of variance followed by Tukey’s multiple comparison test. P-values < 0.05 were considered statistically significant.S1 FigPparg2 (the marker of mature adipocytes) and Cd45 (the marker of leucocytes) mRNA in the adipose tissue MAs and SVF of NC-fed mice by qRT-PCR (n = 3). Mesenteric adipose tissue (a), epididymal adipose tissue (b) and subcutaneous adipose tissue (c). (d−f) Expression of Gpr43 mRNA in the adipose tissue SVF of HFD-fed mice by qRT-PCR (n = 3). Mesenteric adipose tissue (d), epididymal adipose tissue (e) and subcutaneous adipose tissue (f). 18S mRNA expression was used as an internal control. All data are presented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, N.S.: not significant. SVF: stromal vascular fraction.(a−c) Expression of (TIF)Click here for additional data file.S2 Fig+CD11b+F4/80+ cells. Adipose tissue non-macrophage immune cells were sorted by gating on CD45+CD11b-F4/80- cells. We confirmed that the purity of the sorted cells was more than 93%.Adipose tissue macrophages were generated using a cocktail of several antibodies. Adipose tissue macrophage subsets were sorted by gating on CD45(TIF)Click here for additional data file.S3 FigGpr43-/- M2-type bone marrow-derived macrophages. Cells were cultured for 3 h in serum-free medium and stimulated with acetate (10 mM) for 10 min. (b) Quantitative RT-PCR analysis of Tnfα mRNA expression in WT and Gpr43-/- thioglycollate-elicited PECs stimulated for 7 h with acetate (10 mM) (n = 3). 18S mRNA expression was used as an internal control. Body weights (c) and tissue weights (d) of WT and Gpr43-/- mice used in the analysis shown in Gpr43 mRNA expression in LPS-treated  or nontreated Raw264.7 cells. 18S mRNA expression was used as an internal control. All data are presented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, N.S.; not significant. Data are representative of two (a) or three (c) experiments.(a) Effects of acetate (10 mM) on ERK1/2 phosphorylation in WT and (TIF)Click here for additional data file.


The tumor microenvironment (TME) promotes tumor cell invasion and metastasis. An important step in the shift to a pro-cancerous microenvironment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are present in a majority of solid tumors and can directly promote tumor cell motility via cytokine, chemokine and growth factor secretion into the TME. The exact effects that the TME has upon cytoskeletal regulation in motile tumor cells remain enigmatic. The conserved formin family of cytoskeleton regulating proteins plays an essential role in the assembly and/or bundling of unbranched actin filaments. Mammalian Diaphanous-related formin 2 (mDia2/DIAPH3/Drf3/Dia) assembles a dynamic F-actin cytoskeleton that underlies tumor cell migration and invasion. We therefore sought to understand whether CAF-derived chemokines impact breast tumor cell motility through modification of the formin-assembled F-actin cytoskeleton. In MDA-MB-231 cells, conditioned media (CM) from WS19T CAFs, a human breast tumor-adjacent CAF line, significantly and robustly increased wound closure and invasion relative to normal human mammary fibroblast (HMF)-CM. WS19T-CM also promoted proteasome-mediated mDia2 degradation in MDA-MB-231 cells relative to control HMF-CM and WS21T CAF-CM, a breast CAF cell line that failed to promote robust MDA-MB-231 migration. Cytokine array analysis of CM identified up-regulated secreted factors in WS19T relative to control WS21T CM. We identified CXCL12 as a CM factor influencing loss of mDia2 protein while increasing MDA-MB-231 cell migration. Our data suggest a mechanism whereby CAFs promote tumor cell migration and invasion through CXCL12 secretion to regulate the mDia2-directed cytoskeleton in breast tumor cells. In metaproteins . In breaproteins –6. For iproteins . In ordeDuring metastatic dissemination, tumor cells must first escape the primary tumor and invade through the adjacent ECM and TME –10. Tumomammalian diaphanous-related (mDia1-3) formin [The microtubule and actin cytoskeleton are regulated, in part, by the conserved ) formin  family o) formin –17. As m) formin . These s) formin , membran) formin , invadop) formin , 21, and) formin –24.DIAPH1 (encoding mDia1) knockout mice had reduced T cells in the peripheral lymphoid organs and T cell:ECM adhesion and migration were inhibited [DIAPH1 knockout, in conjunction with WASP knockout resulted in defective neutrophil polarization and chemotaxis [mDia formins are regulated through autoinhibition. Autoinhibited mDia proteins exist in a closed or inactive confirmation by an interaction between the Dia inhibitory domain (DID) and the Dia-autoregulatory domain (DAD), which inhibits the functional FH2 domain from associating with actin . Autoinhnhibited , 34. Losemotaxis , 36. Losemotaxis .DIAPH3, increased invasive cell egress from epithelial ovarian cancer spheroids [in vitro and ex vivo [mDia formins were identified as potential therapeutic targets to block tumor cell motility and invasion. Indeed, mDia1 functions in a feedback loop to stimulate mDia1, LARG, RhoA signaling, which in turn modulates cancer cell morphology and invasion . mDia1 wpheroids . Functiopheroids . Convers ex vivo . Thus, tDIAPH3 expression was not diminished in response to CAF-CM treatment. Finally, we determined by membrane-based cytokine array that stromal-secreted CXCL12 is a significantly upregulated component of CAF-CM that underlies mDia2 loss in MDA-MB-231 cells and the resultant increase in cell migration.In this study, we sought to understand how CAF-soluble factors affect the mDia-directed F-actin cytoskeleton in MDA-MB-231 human breast adenocarcinoma cells. Here we demonstrated conditioned media (CM) from WS19T breast tumor-adjacent CAFs significantly increases MDA-MB-231 breast tumor cell migration and invasion, and is correlated with significant loss of mDia2 protein expression through a proteasomal-dependent mechanism. 2 in a humidified environment.MDA-MB-231 breast cancer cells were from ATCC (CRM-HTB-26). Human mammary fibroblasts (HMF) were a kind gift from Dr. Saori Furuta . WS19T and WS21T human breast carcinoma-associated fibroblasts were kind gifts from Dr. Julie Boerner  , and NIHAnti-mDia2, -mDia1, -ROCK1, -β-catenin, and -GAPDH polyclonal rabbit antibodies were from Proteintech and were used at 1:200 dilutions for immunofluorescence and 1:2,000 for western blotting. Anti-RhoA mouse monoclonal antibodies were from Cytoskeleton . Anti-tubulin rabbit polyclonal antibodies were from Abcam . Alexa-Fluor secondary goat anti-rabbit conjugated antibodies were from Invitrogen and were used at 1:200 for immunofluorescence.2O with 16h treatment. Vehicle treatments were equal volumes. SMIFH2 in DMSO  was used at 10–40 μM with 16h for wound closure assays and 8-72h for western blot analysis. Cycloheximide in dH2O was obtained from Sigma-Aldrich and used at 10 μg/ml for 1-24h. CXCL12/SDF-1α in dH2O (R&D Systems) was used at 15–100 ng/ml for 16h for wound closure assays and 8-72h for western blot analysis.Lactacystin (Santa Cruz Biotechnology) was used at 10 μM in dHsiRNA transfections were performed as previously described .Whole cell lysates were collected with lysis buffer , 0.1% bromophenol blue (wt/vol) supplemented with 0.1M diothiothreitol (DTT)) and SDS-PAGE was performed to resolve proteins. Proteins were transferred to PVDF membranes using a BioRad Trans-Blot turbo transfer system. Western blots were exposed using Clarity Western ECL (BioRad) and Alpha Innotech imaging system (Azure Biosystems). Densitometry was performed using ImageJ software.Human mammary, NIH-3T3, WS19T, and WS21T fibroblasts were plated in DMEM growth media, in T-75 culture flasks, and grown to confluence. Media were removed 5 days post-confluence, centrifuged at 1000 rpm to remove cellular debris, and stored at -20°C until use.2. Immediately following scratching, media were changed to either DMEM, HMF-CM, WS19T-CM or WS21T-CM. Image acquisition occurred at introduction of the scratch (0h) and 16h post scratching. “Wounds” were measured using MetaMorph Image Analysis software to determine percent wound closure. Each condition was performed in triplicate within a single experiment and with a minimum of three experimental repeats.Confluent MDA-MB-231 breast cancer cells in 6-well culture plates were scratched with a sterile pipette tip, followed by 16h incubation at 37°C with 5% COSpheroids were generated using centrifugation and poly-HEMA coated, low attachment plates as previously described . Coated Collagen-1 (BD Biosciences) was used at a concentration of 2mg/ml and prepared as previously described (modified from ). 8-well-ΔΔCt). Primers for DIAPH3, PPIB (encoding cyclophilin B), and GAPDH were obtained from Integrated DNA Technologies and used at concentrations following the Radiant protocol. DIAPH3 mRNA levels were normalized to cyclophilin B and GAPDH mRNAs for analysis. Primer sequences were as follows: PPIB (5’-CAT CTG CAC TGC CAA GAC TGA-3’ and 5’-TTG CCA AAC ACC ACA TGC TT-3’), GAPDH (5’- GCC TCA AGA TCA TCA GCA ATG C-3’ and 5’ CCA CGA TAC CAA AGT TGT CAT GG-3’) and DIAPH3 (5’-GCG GGA AAA GGA CTT CAG TAT-3’ and 5’-TCT GTC GGC TTC TCT TAA GAC TTC-3’), and were previously validated and confirmed using BLAST [RNA was isolated using TRIzol (Invitrogen), and cDNA was generated using the Quantitect Reverse Transcription kit (Qiagen) following the manufacturer’s instructions. Quantitative PCR (qPCR) was completed using Radiant SYBR Green Lo-ROX PCR mix  and an Applied Biosystems 7500 PCR system. Analysis was performed on SDS software as previously described [ng BLAST .MTT  cell viability assay was performed following the manufacturer’s specifications. Metabolic activity was assessed at 16, 24, 48, and 72h. Within a single experiment each condition and time point included nine measurements and the experiment was repeated thrice.Sterile coverslips were coated with 10μg/ml of Collagen-1 at 37°C overnight. Cells plated on collagen-coated coverslips were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and stained with a primary antibody against mDia2 overnight at 4°C. Alexa-Fluor 488 secondary antibody (Invitrogen), Alexa-594 phalloidin (Molecular Probes), and DAPI (Invitrogen) were applied at 37°C for 1.5h.Custom cytokine antibody arrays were from RayBiotech . The assA human CXCL12/SDF1-α ELISA kit was obtained from Sigma-Aldrich. The assay was performed following manufacturer’s instructions. The provided standard was performed in triplicate. HMF-CM, WS19T-CM, and WS21T-CM were screened in three independent CM sample collections, collected as previously described. Each collection included parallel HMF-CM, WS19T-CM, and WS21T-CM sample. HMF-CM samples were in duplicate, while WS19T-CM and WS21T-CM were assessed in triplicate. Assays were read at 450nm absorbance using a SpectraMax plate reader and results were plotted with SigmaPlot software.One-tail Student’s t-tests were used with a 95% confidence interval with p < 0.05 interpreted as statistically significant. Standard deviations are shown on histograms.Given the notion that CAF-secreted factors promote invasion and metastasis, we hypothesized that mammary tumor-derived CAFs may influence tumor cell motility through modifying the actin cytoskeleton. We first assessed if factors present in WS19T CAF-conditioned CM altered tumor cell motility in wound healing assays. WS19T CAFs are a tumor-adjacent patient-derived mammary carcinoma associated fibroblast cell line . To asseWe next assessed the specificity of CAF-CM in MDA-MB-231 cell wound closure assays performed for 16h in the presence of CAF-CM or control CM from normal fibroblasts and additional breast tumor-adjacent CAFs. MDA-MB-231 cells and WS19T CAFs alone had 51% and 32% wound closure, respectively, while co-plating MDA-MB-231 cells and (1:1 ratio) resulted in 81% wound closure . HoweverTo assess specificity of CAF-CM upon tumor cell motility, we incubated a panel of normal breast and transformed breast and non-breast tumor cell lines with WS19T-CM. WS19T-CM incubation with MCF-10A and MCF7 cells did not increase MCF-10A motility but did significantly increase MCF-7 motility indicating responsiveness in malignant cells . FurtherTo validate that increased wound closure throughout 16h was not due to increased cell proliferation, we analyzed the effects of WS19T-CM on MDA-MB-231 proliferation using MTT and cell counting assays. Interestingly, WS19T-CM reduced MDA-MB-231 metabolic activity  by 55–85We next assessed whether WS19T-CM affected MDA-MB-231 cell invasion. MDA-MB-231 spheroids were formed for 72h, embedded in 2mg/ml Type-1 collagen gels, and allowed to invade for an additional 72h  Fig 3A)Fig 3A). mDia formins assemble non-branched actin filaments that underlie protrusive structures in motile tumor cells , 53–56. Kinetic washout assays were performed to evaluate if mDia2 loss in response to WS19T-CM was recoverable . MDA-MB-DIAPH3 or control genes encoding cyclophilin B (PPIB) or GAPDH. MDA-MB-231 control and DIAPH3 mRNA levels were assessed after 8-72h of WS19T-CM treatment . HMF-CM was used as a control for baseline levels of these targets in a non-cancerous stromal population and as HMF-CM promoted neither MDA-MB-231 migration nor influenced mDia2 expression. Cytokine targets were chosen based on known CAF-secreted factors and tumor-promoting factors. Cytokines of interest were identified by up-regulation in WS19T-CM arrays relative to WS21T-CM arrays to identify factors with potential to differentially affect MDA-MB-231 cell motility and reduce mDia2 protein expression. TGFα, PDGF, IL-17, TNF-β MMP-13, and CXCL12 all showed dramatically increased expression in the WS19T-CM compared to WS21T-CM .i.e., motility, invasion and metastasis) and the F-actin cytoskeleton. CXCL12, PDGF, and TGFα were further identified as our top three prioritized hits. CXCL12 and its receptor CXCR4 were implicated in cancer cell migration, invasion, and metastasis through formation of chemotactic gradients [Upregulated cytokines were then prioritized based on previous connections to both cancer progression (radients –67. PDGFradients –70 (reviradients ). TGFα iradients , 73.We initially focused upon CXCL12 and its role in promoting MDA-MB-231 invasion and in regulating mDia2 expression. We first evaluated the physiological levels of CXCL12 within our CM panel via CXCL12/SDF1-α ELISA. We assessed CXCL12 levels in HMF-CM, WS19T-CM, and WS21T-CM. Within HMF-CM, WS21T-CM, and WS19T-CM, CXCL12 levels were measured at ~10.5, 31.0, and 136 pg/mL, respectively . We therTo evaluate the effects of CXCL12 upon MDA-MB-231 cell migration, we performed wound closure assays in response to purified CXCL12. We treated MDA-MB-231 cells for 16h with 15, 25, and 100ng/ml CXCL12. CXCL12 treatment resulted in significantly increased wound closure relative to MDA-MB-231 cells in DMEM  . Modest Finally, we evaluated the effects of CXCL12 upon mDia2 expression in MDA-MB-231 cells. MDA-MB-231 cells treated with 100ng/ml CXCL12 for 8-72h had significantly reduced mDia2 protein expression relative to MDA-MB-231 cells in DMEM . Pre-incIn this study, we discovered a role for stromal-derived CXCL12 in breast cancer migration and invasion potentially through proteasome-mediated loss of mDia2 protein expression. WS19T CAF-CM significantly increased motility and invasion of MDA-MB-231 cells relative to MDA-MB-231s in DMEM growth media, or control CM. We observed concomitant and specific loss of mDia2 protein expression with incubation with WS19T CAF-CM. The stromal-derived factor CXCL12 was subsequently identified as a prominent factor enriched in WS19T CAF-CM that promoted, in part, both MDA-MB-231 cell migration, as well as loss of mDia2 protein.DIAPH3 expression is reduced in invasive prostate cancer [DIAPH3 expression was decreased in micro-dissected tumor adjacent stroma derived from invasive breast carcinoma [DIAPH3, or mDia2 expression and/function as a mechanism to control cellular transformation and dissemination may not be employed solely by tumor cells, but by the TME cellular constituency as a whole. It must be noted that this is the first tumor cell model of mDia2 regulation in response to CAF-CM. It warrants expanding studies to additional cancer models where a role for mDia2 in tumor invasion and metastasis has been established, such as prostate, hepatocarcinoma and glioblastoma, to test the specificity of this mechanism of mDia2 dependent mode of motility regulation.We show a loss of mDia2 expression with increased cell migration and invasion in MDA-MB-231 cells in the presence of WS19T-CM. Indeed, we previously demonstrated that mDia2 expression and/or activity was involved in tumor cell motility. Sustained mDia2 activation using small molecule intramics (IMMs) reduced U87 and U251 glioblastoma invasion , while me cancer  and in be cancer . Thus, tarcinoma –77. HencWe examined the effects of the SMIFH2-mediated mDia suppression in our system to evaluate if mDia functional suppression impacted mDia protein stability and/or expression. Indeed, a previous report correlated SMIFH2-mediated functional suppression with loss of mDia2 expression in select cell lines . In our DIAPH3 transcripts are unchanged in the presence of WS19T-CM. We utilized cycloheximide to determine the half-life of mDia2 in culture and compare to WS19T-CM kinetics. The mDia2 half-life in MDA-MB-231 cells treated with CM is significantly shorter than that of cycloheximide-treated cells . Targeting the proteasome with lactacystin both restores mDia2 expression in the presence of CM and blocked the CM-induced increase in cell motility, pointing towards a proteasome-dependent mechanism for loss of mDia2. There is evidence for linkage between mDia2 and the proteasome in tightly regulating mDia2 expression. mDia2, ubiquitin, and the proteasome were shown to directly interact yet mDia2 did not undergo proteasome-mediated degradation in a HEK 293T forced overexpression system [We sought to understand whether the loss of mDia2 was at the level of gene transcription or protein stability/degradation. RT-PCR indicated that levels of n system . Conversn system . Our resWe noted dramatic upregulation of 6 factors by cytokine array analysis of WS19T-CM, relative to HMF-CM and WS21T-CM. We prioritized factors based upon known established links with cancer cell invasion, metastatic formation, cytoskeleton regulation, or mDia formins. From those criteria, CXCL12, PDGF –71, and CXCL12 ELISAs  revealedIn this study, we observed significant increases in breast cancer cell migration and invasion in response to CAF-conditioned media, which was accompanied by dramatic loss of mDia2 expression. We identified CXCL12 as an underlying factor within WS19T-CM that mediates, in part, these phenotypes. Previous work identified a role for exogenous CXCL12 in breast cancer motility and migration, yet the source of CXCL12 influencing tumor motility was uncertain. Here we identify for the first time a physiological source for CXCL12 in the TME- specifically from tumor-adjacent carcinoma associated fibroblasts, and reveal a unique role for the TME in directly influencing tumor cell motility through mDia formin-dependent cytoskeletal regulation. This novel mechanism is a step towards understanding the role of CAF:tumor signaling in cancer progression and identifies potential therapeutic targets that could aid in blocking metastatic dissemination and improving patient prognosis.S1 Table(TIF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 FigA, B. WS19T media was conditioned for 1–5 days prior to collection. WS19T-CM from days 1, 3, and 5 day collections were applied to MDA-MB-231 cells at T0 of a wound closure assay and wounds closed for 16h. Each experiment was performed in triplicate and repeated thrice.(TIF)Click here for additional data file.S2 FigMonolayers of OVCA429, U251, MCF10A or MCF7 cells were wounded and were simultaneously incubated with either control DMEM or WS19T conditioned media for 16 h. Wound closure was measured in triplicate, and the experiment was repeated twice. *p<0.0001 relative to DMEM MCF7 controls.(TIFF)Click here for additional data file.S3 FigA, B. MDA-MB-231 cells plated on glass coverslips were treated with the indicated media for 8h before fixation. Cells were immunostained with anti-mDia2 antibodies, phalloidin and DAPI. Percent nuclear mDia2 fluorescence was measured relative to plasma membrane/cytoplasmic mDia2 fluorescent signal with Metamorph software. At least 30 cells per condition were measured and the experiment was repeated three times. Scale bars = 25μm.(TIF)Click here for additional data file.


Poor water-solubility of artesunate (ARS) hampers its clinical application. We here covalently linked ARS to PEGylated nanographene oxide (nGO-PEG) to obtain ARS-modified nGO-PEG (nGO-PEG-ARS) with excellent photothermal effect and dispersibility in physiological environment. nGO-PEG-ARS induced reactive oxygen species (ROS) and peroxynitrite (ONOO─) generations. Although nGO-PEG with near-infrared (NIR) irradiation did not induce cytotoxicity, the photothermal effect of nGO-PEG under NIR irradiation enhanced not only cell uptake but also ONOO─ generation of nGO-PEG-ARS, resulting in the synergistic chemo-photothermal effect of nGO-PEG-ARS in killing HepG2 cells. Pretreatment with Fe(III) 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato chloride  instead of antioxidant N-Acetyle-Cysteine  significantly blocked the cytotoxicity of nGO-PEG-ARS with or without NIR irradiation, demonstrating that ONOO─ instead of ROS dominated the synergistic chemo-photothermal anti-cancer action of nGO-PEG-ARS. nGO-PEG-ARS with NIR irradiation resulted in a complete tumor cure within 15 days earlier than other treatment groups, and did not induce apparent histological lesion for the mice treated with nGO-PEG-ARS with or without NIR irradiation for 30 days, further proving the synergistic chemo-photothermal anti-cancer effect of nGO-PEG-ARS. Collectively, nGO-PEG-ARS is a versatile nano-platform for multi-modal synergistic cancer therapy. Physi in vivo –11. More in vivo –16.π-π stacking) between aromatic molecules and aromatic regions of nGO [π-π stacking and hydrophobic interactions [in vivo and in vitro [π-π and hydrophobic interactions and exhibiting dramatically synergistic anti-cancer effect of PTT and chemotherapy in vitro and in vivo [Nano-graphene oxide (nGO) is a potential nano-platform for chemo-photothermal synergistic therapy –20. Highs of nGO –25. Yangractions . Liu andractions . Zhang ain vitro . We rece in vivo .Artesunate (ARS) and dihydroartemisin (DHA), the first line anti-malarial drugs with only minimal side-effects, possess profound anticancer activity against various cancer cell lines –31. Howe─) not reactive oxygen species (ROS) dominated the synergistic chemo-photothermal anti-cancer effect of nGO-PEG-ARS. In addition, the mild photothermal effect of nGO-PEG by NIR irradiation significantly enhanced cell uptake and ONOO─ generation of nGO-PEG-ARS, contributing to the outstanding synergistic chemo-photothermal anti-cancer effect of nGO-PEG-ARS, which was verified by in vivo experiments in Balb/c mice bearing 4T1 murine breast cancer tumors.In this report, we developed an ARS-loaded nGO-PEG composite (nGO-PEG-ARS) for synergistic chemo-photothermal therapy of cancer via two steps Scheme : 1) cova2 groups of PEG, whereas the zeta potential of nGO-PEG-ARS increased to -17.6 mV due to the linkage of ARS  compared with nGO (-45.4 mV) due to the reaction between the negative carboxyl groups of nGO and the NHS Figure . As show-1), C=O (∼1726 cm-1), C=C (∼1575 cm-1) and C-O (∼1059 cm-1) are the functional groups of the carboxylated nGO [-1) and C-O-C (∼1100 cm-1) stretching vibrations are the functional groups of PEG [-1 (-CH2) and strong stretching vibration presented at ∼1100 cm-1 (C-O-C)  Figure  demonstr) Figure , which mPEGylation was designed to impart the high degree of hydrophilicity for nGO . In ordeExcellent water-solubility of nGO-PEG-ARS can not only retain the biological activity of ARS but also effectively reduce the dose of intravenous injections due to the potentially prolonged duration of nGO-PEG-ARS , 45. nGO2 for 5 min. After NIR irradiation for 5 min, control water had no response to NIR irradiation, whereas the temperature of nGO-PEG/nGO-PEG-ARS solutions increased to ∼35 °C (40 μg/mL), 44 °C (80 μg/mL) and 60 °C (120 μg/mL)  were exposed to the 808 nm NIR laser at a power density of 2 W/cm) Figure , which w) Figure .It has been demonstrated that mild photothermal heating to ∼43°C of nanoparticles by lower power NIR laser irradiation was able to enhance the intracellular delivery of chemotherapy drugs or photosensitizers for improved cancer cell killing , 47. To 2 for 3 min  of nGO-PEG or nGO-PEG-ARS for 48 h in the presence or absence of NIR irradiation. The nGO-PEG-ARS group had an equivalent nGO dosage to the nGO-PEG group. Both the nGO-PEG group and nGO-PEG-ARS groups were irradiated by NIR light  in the presence or absence of NIR irradiation. ARS induced a dose-dependent cytotoxicity, which was not enhanced by NIR irradiation Figure , suggestWe recently demonstrated that ROS was not involved in ARS-induced apoptosis in HepG2 cells . FCM ana─ from the reaction between NO and O2·− dominated the sodium nitroprusside (SNP)-induced apoptosis in HepG2 cells [─ might dominate the synergistic chemo-photothermal anti-cancer effect of nGO-PEG-ARS. As expected, FCM analysis with DHR 123 staining showed that nGO-PEG-ARS treatment for 6 h potently induced abundant ONOO─ generation which was significantly enhanced by NIR irradiation  for 2 h completely inhibited the cytotoxicity of nGO-PEG-ARS and significantly inhibited the cytotoxicity of nGO-PEG-ARS plus NIR irradiation  were cultured at 50 °C for 15 min before FCM analysis, exhibiting that 50 °C significantly enhanced ARS-induced NO . N-hydroxysuc-cinimide (NHS) and N-(3-(dimethylamino)propyl-N′-ethylcarbodiimide) hydrochloride (EDC) were purchased from Sigma-Aldrich . 8-Armpolyethylene glycol-amine  was purchased from Seebio Biotech Inc. . ARS was obtained from Chongqing Holley Pharmaceutical Co., Ltd. . Fetal bovine serum (FBS) was purchased from Sijiqing . Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Thermo Scientific (USA). Roswell Park Memorial Institute 1640 (RPMI 1640) was purchased from Gibco (Grand Island).2) (10 kDa) via amido bond using EDC chemistry for 12 h, and the PEGylated nGO  was then subsequently covalently functionalized with ARS by EDC/NHS activation reaction to synthetize nGO-PEG-ARS.GO was obtained by oxidation of graphite following the modified Hummers method . Nanosiz-1 under N2 flow. The images of all samples  were recorded using a digital camera .The sizes and zeta potentials of nGO, nGO-PEG and nGO-PEG-ARS were confirmed by Zetasizer Nano ZS . UV-vis spectra were performed by a UV-vis spectrometer  with a 1 cm quartz cuvette. Fourier transfer infrared (FT-IR) spectra were recorded on a Nicolet 6700 FT-IR spectrometer . Thermogravimetric analyses (TGA) were performed on a thermogravimetric analyzer  from room temperature to 700 °C at a heating rate of 5 °C min5 cells/well and incubated with 80 μg/mL  of FITC/nGO-PEG-ARS for 2, 4 and 8 h with or without NIR irradiation respectively in 1.0 mL DMEM medium containing 10% fetal bovine serum, and then, the cells were rinsed and collected by phosphate buffered saline (PBS). The uptake ratio of nGO-PEG-ARS by HepG2 cells was measured by flow cytometry  using FITC labeled on nGO-PEG-ARS, and 10,000 events were recorded for each FCM analysis.The nGO-PEG-ARS was labeled by fluorescein isothiocyanate . In brief, the solution of nGO-PEG-ARS  was mixed with 0.1 mL FITC (13 mM) dissolved in DMSO and then stirred over night at room temperature. The resulting mixtures were filtrated through 30 kDa filters Millipore and centrifuged at 6500 × g to remove aggregated FITC, giving water-solubility FITC/nGO-PEG-ARS. The whole procedures were operated in dark place. HepG2 cells were plated into 6-well plates at a density of 1 × 102 for 5 min. The temperatures of water, nGO, nGO-PEG and nGO-PEG-ARS solutions were measured by a thermocouple thermometer  every other 30 s. In addition, the thermal images of these solutions were also measured by an infrared thermal camera . All experiments were conducted at room temperature.The test solution (1 mL) was introduced in an eppendorf tube (1.5 mL) and exposed to the 808 nm laser irradiation at a power density of 2 W/cm4 cells/well with 100 μL of medium and incubated for 24 h. After removing DMEM medium, the cells were exposed to 40, 80 and 120 μg/mL of nGO-PEG or nGO-PEG-ARS respectively for 4 h in 100 μL of media containing 10% fetal bovine serum, and then irradiated with 2 W/cm2 of 808 nm NIR laser for 3 min or 2 W/cm2 of 808 nm NIR laser for 5 min followed by further incubation for 44 h. Cell viability was evaluated by using Cell Counting Kit-8  assay as described previously [HepG2 cells were cultured in a DMEM medium containing 10% fetal bovine serum at 37 °C in a humidified 5% CO2 atmosphere. For cell toxicity assay, HepG2 cells were plated into 96-well plates at a density of 1 × 10eviously . Results+/PI−(early apoptotic cells) and Annexin V-FITC+/PI+ (late apoptotic cells).Cell apoptosis was quantified by FCM analysis using Annexin V-FITC/PI apoptosis detection kit  according to the manufacture's protocol as previously described . ApoptotΔΨm) was analyzed by FCM assay using ΔΨm-specific stain Rho123 as previously described [ΔΨm.Characteristic breakdown of mitochondrial transmemberane potential  and Dihydrorhodamine 123  as described previously [─ to the highly fluorescent product rhodamine. HepG2 was cultured with nGO-PEG and nGO-PEG-ARS for the indicated times with or without 808 nm NIR laser irradiation (5 W/cm2) for 15min, and then the cells were stained with 20 μM DCF-DA in PBS for 30 min or with 10 μM DHR 123 for 20 min in the dark at 37 °C. Then the cells were harvested and washed twice with PBS before FCM analysis.ROS and ONOOeviously . DCF-DA 2 of 808 nm NIR laser for 15min, and then the cells were stained with 5 μM DAF-FM DA for 30 min at 37 °C. After washing with PBS three times, the samples were analyzed by FCM analysis.Intracellular NO level was quantified by using FCM analysis with DAF-FM DA staining as described previously . HepG2 c6 cells in 200 mL PBS into the flank region of Balb/c mice. The dimension of tumors was monitored by digital calipers. After tumor size reached approximately 60 mm3, the mice were randomized into 6 different treatment groups (5 mice per group): control group, nGO-PEG group, nGO-PEG-ARS group, NIR group, nGO-PEG plus NIR group and nGO-PEG-ARS plus NIR group. Balb/c mice bearing 4T1 tumors in different groups were intratumorally injected with nGO-PEG, nGO-PEG-ARS , respectively. For NIR irradiation groups, after 2 h injection, the tumor region of mice was irradiated by 808 nm NIR laser for 15 min. During NIR irradiation, full-body thermographic images were captured using an infrared camera . After treatment, all mice were returned to animal housing and tumor volumes were tracked every 3 days by digital caliper measurements. The tumor volume was calculated by formula V = (longest dimension) ∙ (shortest dimension)2/2. Control and nanocomposites-treated mice were sacrificed 30 days after treatment. Major organs of those mice were collected, fixed in 4% formalin, conducted with paraffin embedded sections, stained with hematoxylin and eosin (H&E), and examined under a digital microscope.Female Balb/c mice (4-6 weeks old) were obtained from Laboratory animal center of Sun Yet-sen University and approved by Guangdong Province Experimental Animals Monitoring Institute. 4T1 tumor bearing mice were established by subcutaneous injection of 5 × 10in vitro and in vivo. ONOO─ dominated the synergistic chemo-photothermal anti-cancer effect of nGO-PEG-ARS. The photothermal effect of nGO-PEG under NIR irradiation significantly enhanced not only cell uptake but also ONOO─ generation of nGO-PEG-ARS, contributing to the outstanding synergistic chemo-photothermal anti-cancer ability of nGO-PEG-ARS.In summary, we successfully developed an ARS-loaded nGO-PEG composite (NGO-PEG-ARS) by covalently linking ARS to PEGylated nGO for synergistic chemo-photothermal therapy of cancer. nGO-PEG-ARS exhibited excellent dispersibility in physiological environments and synergistic chemo-photothermal anti-cancer effect


Caenorhabditis elegans, the nuclear receptor DAF-12 acts as a dietary and environmental sensor to orchestrate diverse aspects of development, metabolism, and reproduction. Recently, we have reported that DAF-12 together with co-repressor DIN-1S is required for starvation tolerance by promoting fat mobilization. In this report, we found that genetic inactivation of the DAF-12 signaling promoted the production of reactive oxygen species (ROS) during starvation. ROS mediated systemic necrosis, thereby inducing organismal death. The DAF-12/DIN-1S complex up-regulated the expression of antioxidant genes during starvation. The antioxidant enzyme GST-4 in turn suppressed ROS formation, thereby conferring worm survival. Our findings highlight the importance of antioxidant response in starvation tolerance and provide a novel insight into multiple organisms survive and adapt to periods of nutrient deprivation.Animals often experience periods of nutrient deprivation; however, the molecular mechanisms by which animals survive starvation remain largely unknown. In the nematode  Caenorhabditis elegans can make physiological changes to developmentally arrest at multiple stages, such as L1 diapause4fil-1 and fil-2 that involved in converting fat storage into energy, and thus maintain whole-body energy homeostasislipl-1 and lipl-3, is up-regulated by a transcription factor HLH-30All animals have evolved abilities to improve the chances of survival and reproduction. Among these stress, starvation is one of common stressful situations in nature. To cope with nutrient deprivation, the free-living nematode C. elegans, the nuclear receptor DAF-12 orchestrates a switch from arrest to developmental progression in response to environmental and dietary cues, and has been implicated as a signal connecting nutrition, development, and longevity66tbh-1 that encodes tyramine β-hydroxylase, a key enzyme for octopamine biosynthesis in C. elegans. Octopamine up-regulates the expression of the lipase gene lips-6 in the intestineIn daf-12 and din-1 mutants were significantly lower than that of tbh-1 mutants. It is possible that in addition to the octopamine pathway, there is another pathway through which DAF-12/DIN-1S acts to regulate starvation resistance. A previous microarray analysis has revealed that expression of antioxidant and detoxification genes is up-regulated during starvation in C. elegansdaf-12(rh61rh411) or din-1(dh127) resulted in an increase in reactive oxygen species (ROS) formation, which was involved in worm death, after starvation.We noted that the survival rates of mir-84p::GFP and mir-241p::GFP. Both the microRNAs are the targets of liganded DAF-12, and their expressions were downregulated by mutations in daf-12 and daf-9mir-84p::GFP and mir-241p::GFP was significantly lower in worms after 12 h starvation than in worms fed with the standard laboratory food E. coli OP50 (mir-84 and mir-241 by qPCR (daf-9 mutant worms) are more stress resistant in a manner dependent on unliganded DAF-12 (the DAF-12/DIN-1S complex)daf-12(rh611rh411) or din-1(dh127) abolished the stress resistance-phenotype of starved worms. These results further confirm this notion that the DAF-12/DIN-1S complex is dominant during nutrient deprivation.It is believed that DAF-12 is mostly unliganded under starvation conditions, whereas production of DAs by DAF-9 converts DAF-12 to a liganded state when food is available6oli OP50 . Similar by qPCR . DA-defitbh-1 expression in response to starvation in C. elegans. The DAF-12/DIN-1S-TBH-1 -octopamine signaling is required for starvation tolerance. However, more than 60% of daf-12(rh61rh411) or din-1(dh127) mutants were dead (tbh-1(n3247) worms were dead after 5 days of starvation. These results suggest that in addition to octopamine, other mechanisms exist for DAF-12/DIN-1S to exert its effect on starvation resistance.Our recent study has revealed that the DAF-12/DIN-1S complex induces ere dead , whereasC. elegans, nutrient deprivation induces apoptosis in germ cells and necrotic cell death in neurons12daf-12(rh61rh411) or din-1(dh127) mutants. Using the SYTO 12 dye staining against the apoptotic germ cellsdaf-12(rh61rh411), din-1(dh127), and WT worms after two days of starvation  and din-1(dh127) mutants after five days of starvation (In arvation . Meanwhiarvation . Therefodaf-12(rh61rh411), din-1(dh127), and tbh-1(n3247) mutants displayed an enlarged vacuolar morphology characteristic of necrotic cells in the head and intestine, whereas WT worms had fewer vacuolated cells after two days of starvation (daf-12(rh61rh411), din-1(dh127), and tbh-1(n3247) mutants, but not in WT worms, after two days of starvation  images showed that arvation . Furtherarvation . Similararvation . These rC. elegans, many genes involved in necrosis have been identified, including calpains and aspartyl proteases17clp-1, clp-2, tra-3/clp-5, clp-6, clp-7. Organismal death was significantly suppressed in daf-12(rh61rh411) or din-1(dh127) mutants subjected to RNAi with tra-3/clp-5  or din-1(dh127) mutants upon starvation (asp-1 to asp-6) in C. elegans. We found that inhibition of asp-4 by RNAi, but not other aspartyl proteases , UDP-glucuronosyltransferases , and cytochrome P450 (cyp-35D1 and cyp-29A3), is up-regulated during starvationC. elegans19gst-4 and gst-10 was significantly up-regulated in WT and tbh-1(n3247) worms, but not in daf-12(rh61rh411) or din-1(dh127) worms, during one day of starvation  worms, but not in daf-12(rh61rh411) or din-1(dh127) worms  mutants (It is well established that the DAF-12/DIN-1S complex mediates resistance to heat and oxidative stressarvation . Using t7) worms . Thus, t mutants . These r2DCFDA), a fluorescent dye that has been used to detect the ROS levels in C. elegans. As shown in daf-12(rh61rh411) or din-1(dh127) mutants following starvation. It should be noted that a mutation in daf-12(rh61rh411) and din-1(dh127) itself did not lead to an increase in ROS levels under well-fed conditions. Similar results were obtained using fluorescent dyes, dihydroethidium (DHE) and CellROX® Deep Red  and din-1(dh127) mutants after 12 h of starvation (daf-12(rh61rh411) and din-1(dh127) mutants . To test this idea, we first determined the levels of ROS using 2′,7′-dichlorodihydrofluorescein diacetate (HDeep Red  and c. Larvation  and S6. arvation . Meanwhi mutants  and c. Tdaf-12(rh61rh411) or din-1(dh127) mutants, worms were treated with N-acetylcysteine (NAC) (1 mM), a ROS scavengerdaf-12(rh61rh411) or din-1(dh127) mutants after 24 h starvation (daf-12(rh61rh411) and din-1(dh127) mutants , and α-tocopherol, also suppressed the ROS formation in the starved worms subjected to t-4 RNAi  and S6. C. elegans. After nutrient deprivation, the liganded DAF-12 (DAF-12/DA) shifts the equilibrium to ligand-free DAF-12/DIN-1S complex. DAF-12/DIN-1S in turn boosts the antioxidant response, which maintains redox homeostasis. Downregulation of this pathway exacerbates oxidative stress, thereby mediating nutrient deprivation-induced organismal death.Our studies uncover a surprising role for the antioxidant response in starvation resistance in C. elegans, which is a non-feeding alternative larval stage2324daf-12 or din-1 significantly elicits ROS formation during starvation supports this idea.Up-regulation of antioxidant and detoxification genes is widely observed in a variety of organisms under nutrient deprivation conditions. A microarray analysis reveals that a set of stress resistance and detoxification related genes is up-regulated during starvation in adult wormsC. elegans and micegst-4 and gst-10 and. Knockdown of gst-4 increases the abundance of ROS, and worm death during starvation. More importantly, suppression of ROS by NAC partially rescues worm death in daf-12(rh61rh411) or din-1(dh127) mutants. These results suggest that the DAF-12/DIN-1S complex confers starvation resistance, at least in part, by a mechanism of its action involved in the antioxidant defense. To counter against elevated ROS-induced tissue injury, compensatory antioxidant response is crucial for starvation tolerance. Clearly, the mechanism underlying the DAF-12/DIN-1S complex-mediated antioxidant response needs to be investigated further in light of our current results.Starvation response protects mice and yeast against oxidative stress28daf-12(rh61rh411) and din-1(dh127) mutants. Genetic inactivation of components in the necrosis pathway  can delay worm death, confirming a role for systemic necrosis in starvation-induced organismal death. Although we tested the effect of five calpains and six aspartyl proteases on worm death, however, only knockdown of tra-3 and asp-4 by RNAi inhibits worm death. In this study, we tested the knockdown efficiency of RNAi on knockdown of all of the calpains and aspartyl proteases, and found these genes expressions were significantly ablated by RNAi. However, we really do not know why only knockdown of tra-3 and asp-4 displays a significant protection. As the antioxidant NAC significantly suppresses systemic necrosis in daf-12(rh61rh411) and din-1(dh127) mutants during starvation, ROS is likely to be involved in systemic necrosis.At present, the mechanisms underlying death of the whole organism after nutrient deprivation remain incompletely understood. Although apoptosis occurs in germ cells of worms after nutrient deprivation12C. elegans that DAF-12/DIN-1S, which is activated during starvation, up-regulates antioxidant genes. Induction of antioxidant responses clears the detrimental effects of ROS on worm tissues, and ultimately promotes survival during starvation. Increased antioxidant response seems to be a common phenomenon among many organisms during nutrient deprivation. Thus, dysregulation of antioxidant response contributes to cell death in the process.In summary, our results demonstrate in C. elegans strains were cultured under standard conditions and fed E. coli OP50C. elegans Bristol N2. Mutated strains used in this stud, including daf-12(rh61rh411), din-1(dh127), tbh-1(n3247), and strains containing mir-84p::GFP, mir-241p::GFP, and Pgst-4::gst-4::GFP, were kindly provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).The The clones of genes for RNAi were from the Ahringer libraryEscherichia coli OP50. Well-fed young adult worms in plates containing 10 mM menadione, and E. coli OP50 were used as control. For heat stress, 40–50 young adult worms were transferred to plates in the absence of E. coli OP50 at 35 °C. Well-fed young adult worms in the presence of E. coli OP50 were used as control. The number of living worms was counted at 5 h intervals until all of the worms were dead. Immobile worms unresponsive to touch were scored as dead. Three plates were performed per assay and all experiments were performed three times.After synchronized young adult worms were starved for 12 h, the worms were transferred to plates containing 10 mM menadione  in the absence of standard food E. coli OP50 at 20 °C until the young adult stage. 40–50 worms were then transferred to NGM agar plates containing 5′-fluoro- 2′-deoxyuridine (FUdR) (75 μg/ml), ampicillin (100 μg/ml), kanamycin (50 μg/ml), and amphotericin-B (0.25 μg/ml) in the presence or absence of E. coli OP50 at 20 °C. The number of living worms was counted at five days. Immobile worms unresponsive to touch were scored as dead. Three plates were performed per assay and all experiments were performed three times.Synchronized populations of L1 larva were cultivated on NGM plates in the presence of After worms were starved for two days, the worms were staining with M9 medium containing 20 μM SYTO 12 green (Invitrogen) for 90 minAfter worms were starved for two days, the worms were stained in M9 medium containing 1 mM acridine orange mir-84p::GFP, mir-241p::GFP, and Pgst-4::gst-4::GFP analysis, synchronized young adult worms were starved for 12 h. Then the worms were mounted in M9 onto microscope slides. The slides were imaged using a Nikon e800 fluorescence microscope. Fluorescence intensity was quantified by using the ImageJ software (NIH). Mean value and standard errors were calculated based on more than 100 worms under each condition in three independent experiments.For ® Premix-Ex TagTM  on a Roche LightCycler 480® System . The primers used for PCR were as follows: gst-4: 5′-TGG AGA CTC ATT GAC TTG GG-3′ (F), 5′-TCC TTT CTT GTT GCC ACG-3′ (R); gst-10: 5′-CGT GCC ACA ACT TTA CTA CTT C-3′ (F), 5′-CAA CTG ACC AAG GAG CAT TC-3′(R); act-1: 5′-GGG CGA AGA AGG AAA TGG TC-3′ (F), 5′-CAG GTG GCG TAG GTG GAG AA-3′ (R).Total RNA was isolated from worms with TRIzol Reagent (Invitrogen). Random-primed cDNAs were generated by reverse transcription of the total RNA samples with SuperScript II (Invitrogen). A real time-PCR analysis was conducted using SYBR® Deep Red Reagent, respectively. Briefly, after 24 h of starvation, worms were incubated with 3 μM of DHE in M9 medium for 30 min. Then worms were washed three with PBS, and mounted in M9 onto microscope slides. For ROS detection using CellROX® Deep Red Reagent, worms were fixed by 2% of paraformaldehyde for 30 min. After washed with M9 medium for three times, the worms were incubated with 5 μM of CellROX® Deep Red Reagent for 1 h. Then worms were washed three with PBS, and mounted in M9 onto microscope slides. The slides were imaged using a Nikon e800 fluorescence microscope. Mean value and standard errors were calculated based on more than 100 worms under each condition in three independent experiments.After starvation for 24 h, the ROS levels were detected by 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) as a probe as described previouslyThe statistical significance of differences in gene expression, starvation survival, and fluorescence intensity was assessed by performing a one-way ANOVA followed by a Student-Newman-Keuls test. Differences in survival rates for oxidative/ heat stress treatment were analyzed using the log-rank test. Data were analyzed using SPSS17.0 software (SPSS Inc.).How to cite this article: Tao, J. et al. Antioxidant response is a protective mechanism against nutrient deprivation in C. elegans. Sci. Rep.7, 43547; doi: 10.1038/srep43547 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Staphylococcus aureus sequence type 398 (ST398) is a rapidly emerging livestock-associated strain causing zoonotic disease in humans. The course of pathogen evolution remains unclear, prompting whole-genome comparative studies in attempts to elucidate this issue. We present the full, annotated genomes of five newly isolated representative ST398 strains from five major sequence heterogeneity groups of our diverse isolate collection. Staphylococcus aureus multilocus sequence type (MLST) ST398 was originally reported as a livestock-associated methicillin-resistant S. aureus (LA-MRSA) (3LA-MRSA) , 2. SincA-MRSA) 36. The anA-MRSA) 3, 8. We aThe genomes were sequenced with Pacific Biosciences (PacBio) RSII sequencing technology, using one single-molecule real-time (SMRT) cell. Contig assembly was performed using the Hierarchical Genome Assembly Process (HGAP) workflow –11 and g–CP019591; GD5, CP019592; GD705, CP019593; GD1539, CP019594; and GD1677, CP019595.The complete genome sequences have been deposited in GenBank with accession numbers as follows: 293G,


Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1β production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during   •IL-4-activated STAT6 acts as a transcriptional repressor in macrophages•IL-4-STAT6-repressed enhancers associate with reduced LDTF and p300 binding•Inflammatory responsiveness of the IL-4-repressed enhancers is attenuated•IL-4 limits the LPS-induced inflammasome activation, IL-1β production, and pyroptosis The molecular bases of repressive transcriptional mechanisms contributing to macrophage polarization are not well understood. Czimmerer et al. show that in alternatively polarized macrophages, IL-4-activated STAT6 represses a large set of enhancers modulating the transcriptional program. STAT6-repressed enhancers are characterized by reduced chromatin accessibility, eRNA expression, LDTF, and p300 binding. IL-4-STAT6-mediated repression limits the inflammatory responsiveness including inflammasome activation, IL-1β production, and pyroptosis. Thus, the IL4-STAT6 pathway establishes an epigenomic signature to selectively repress the macrophage inflammation program. Macrophage plasticity is ensured by dynamic and partially reversible responsiveness to pathogen-derived molecules as well as the cytokine and lipid microenvironment. The two well-characterized extreme functional outcomes of macrophage polarization are T helper 1 (Th1) cell-type cytokine interferon-gamma (IFN-γ)-induced classical or M(INF-γ)-type polarization with enhanced bactericidal capacity and Th2 cell-type cytokine interleukin-4 (IL-4)-induced alternative or M(IL-4)-type polarization with anti-inflammatory properties, but complex molecular cues can generate an entire spectrum of different activation states .The major determinant of macrophage plasticity is their specific transcriptional program dictated primarily by lineage-determining transcription factors (LDTFs) including ETS-domain transcription factor PU.1, CCAAT/enhancer binding proteins (C/EBPs), activator protein 1 (AP-1), or Runt-related transcription factor 1 (RUNX1) as well as extracellular signal-dependent transcription factors (SDTFs) including LPS-activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) or AP-1, IFN-γ-activated signal transducer and activator of transcription 1 (STAT1), or IL-4- and IL-13-activated STAT6; for a review see in vitro modeling of complex immunological microenvironment by IL-4 and IFN-γ co-stimulation leads to the attenuation of IFN-γ-induced transcriptional activation due to the effects of IL-4 on restrictive set of auxiliary transcription factors in mouse macrophages  using RNA-seq  and three activated  genes with RT-qPCR, and we confirmed the IL-4-mediated and STAT6-dependent regulation (We determined the STAT6-dependent IL-4-regulated genes in a time course in wild-type (WT) and  RNA-seq A. First, RNA-seq . We also RNA-seq . IL-4-me RNA-seq B. For vagulation C and S1CYm1 and Fizz1/RELM-α  and thioglycollate-elicited peritoneal macrophages (Thio-Mac) utilizing publicly available RNA-seq data  among the genes that were downregulated in response to nematode infection in peritoneal macrophages , the active histone mark H3K27Ac using chromatin immunoprecipitation sequencing (ChIP-seq), and nascent RNA expression by Global Run-On sequencing (GRO-seq) after 1 hr of exposure. Elongation-specific RNAPII-pS2 ChIP-seq revealed 5,931 gene bodies, exhibiting significantly changing read enrichments  . RNAPII-We also determined the STAT6 cistrome using a time course of 1, 6, and 24 hr of IL-4 stimulation A. STAT6 Next we assigned STAT6-bound genomic regions to genes in order to assess the correlation between IL-4-repressed enhancer activity (RNAPII-pS5 by ChIP-seq) and gene expression (mRNA by RNA-seq). For this analysis, we predicted the sub-topologically associated domains (subTADs) in which gene regulation by STAT6 might take place, using CTCF and RAD21 ChIP-seq datasets from BMDM, utilizing a previously described algorithm . As showTo understand the IL-4-STAT6 signaling-mediated transcriptional regulation in more detail, we carried out analyses on individual genes and enhancers. For the selected repressed and activated genes, we identified at least one STAT6-bound enhancer showing reduced and induced H3K27 acetylation and RNAPII binding, respectively F. EnhancIn order to investigate whether the functional characteristics of STAT6 peaks (activator versus repressor) are influenced by their genomic localization and/or the DNA sequences they are associated with, we analyzed the genomic distribution of STAT6 peak clusters. We found only minor differences between the distinct STAT6 peak clusters regarding genomic localization relative to genes . The majde novo motif enrichment analysis of the sequences under the STAT6 peaks. PU.1, TRE, RUNX, and C/EBP motifs were enriched under all three clusters  experiments. Our genome-wide analyses showed increased chromatin accessibility at the activator STAT6-bound sites A, while Chromatin openness determines enhancer activity in different cell types . MoreoveThe acetylation status and thus the activity of enhancers are tightly controlled by histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes . Therefoin vitro and in vivo . We selected four genes for this analysis  based on their IL-4-STAT6-HDAC3-dependent repression  was partially attenuated by IL-4-STAT6 signaling pathway similarly to Fcgr1_−7.7Kb and Il18_+25Kb enhancers described above  and injected LPS or Salmonella Typhimurium into the peritoneal cavity 9 days after nematode infection. As expected, the number of the M2 macrophage marker Ym1-positive macrophages was highly induced in peritoneal macrophages of H. polygyrus-infected mice, confirming alternative macrophage activation following nematode infection  without direct DNA binding or (2) by recognizing non-canonical STAT6-binding motifs. It remains to be identified which mechanism is at play and, if STAT6 binding to the DNA is indirect, which DNA-bound factor interacts with STAT6. Our motif analysis suggests that the lineage-determining factors (PU.1 and C/EBPα) are the most likely candidates though PU.1 and C/EBPα binding also decreased at STAT6-repressed enhancers. Thus this requires further investigations.Although the global transcriptional repressor activity of the key TFs for macrophage polarization  is not fully understood, several other SDTFs have been shown to possess distinct repressor activities. Ligand-activated nuclear receptors including PPARs and LXRs are able to reduce the activity of inflammatory SDTFs through transrepression carried out by direct protein-protein interactions without direct DNA binding . It has STAT6-mediated repression appears to be distinct from other repressive mechanisms. Histone acetylation and gene expression tightly depends on the cofactor composition (HAT:HDAC ratio) . SeveralBrugia malayii-implanted mice compared to thioglycollate-elicited macrophages. This formally suggests that alternative polarization likely induces partial desensitization of macrophages to further inflammatory signals in vivo. Accordingly, M2-type macrophages have been shown to protect mice against chemically induced colitis and a growing body of evidence indicates that clinically controlled helminth infection is able to ameliorate inflammatory bowel disease (IBD)  .These studies suggest that complex bidirectional interactions exist between different polarization signals that determine the overall sensitivity and responsiveness of macrophages toward environmental stimuli. Our findings provide insights into this cross-talk at the level of individual enhancers and raise the intriguing possibility that IL-4-STAT6 signaling, through direct transcriptional repression of inflammatory enhancers, induces desensitization of macrophages to microbial-, stress-, and damage-associated endogenous signals.lnagy@sbpdiscovery.org).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Laszlo Nagy . Following this step second strand cDNA synthesized, double stranded cDNA end repaired and 3′ ends adenylated then Illumina index adapters were ligated. After adaptor ligation enrichment PCR was performed to amplify adaptor ligated cDNA fragments. Fragment size distribution and molarity of libraries were checked on Agilent BioAnalyzer DNA1000 chip . Paired read 100bp sequencing runs were performed on Illumina HiScan SQ instrument .cDNA library for RNA-Seq was generated from 1 μg total RNA using TruSeq RNA Sample Preparation Kit  according to the manufacturer’s protocol. Briefly, poly-A tailed RNAs were purified by oligodT conjugated magnetic beads and fragmented on 94 C degree for 8 minutes, then 1ChIP was performed essentially as previously described . LibrariATAC-seq was carried out as described earlier with minor modification . Cells wGRO-seq was performed as described earlier , but thePpia. Primer sequences are available in RNA was isolated with Trizol reagent (Ambion). RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer’s protocol. Transcript quantification was performed by qPCR reactions using SYBR green master mix (BioRad). Transcript levels were normalized to LDH activity was measured in the supernatants of unstimulated and IL-4-pretreated WT and STAT6KO bone marrow-derived macrophages after IL-4 pretreatment and/or LPS/ATP costimulation (LPS-exposed BMDMs were treated with ATP for 30 min) by commercially available LDH UV assay on Cobas c 501 instrument . This measurement is based on the conversion of L-lactate to pyruvate along with the reduction of NAD+ to NADH. The initial rate of the NADH formation was directly proportional to the catalytic LDH activity determined by photometrically measuring the absorbance increment at 340 nm.LPS-exposed BMDMs were treated with ATP for 45 min. Supernatants from ATP-treated macrophages were collected, centrifuged and stored at −20°C until further use. IL-1β was measured from samples using ELISA kit  according to the manufacturer’s instructions and analyzed on FlexStation 3 Microplate Reader (Molecular Devices). The minimum detectable dose is 15.6 pg/ml.Cells were harvested and centrifuged, then they were lysed in loading buffer . Before loading all samples were boiled for 10 minutes. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were then blocked with 5% non-fat milk, washed briefly, incubated with primary antibodies at 4°C overnight. Pro-IL-1β (AF401-NA) was from R&D System, ASC (sc22514-R) was from Santa Cruz, pro-caspase-1 (AG-20B-0042) and NLRP3 (AG-20B-0014) antibodies were obtained from AdipoGen. Primary antibodies were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies from BioRad for 1 hour at room temperature. Proteins were visualized by Supersignal West-Pico peroxide/luminol enhancer solution from Pierce. To verify the loading of equal amount of protein sample, the β-actin (Sigma-Aldrich) expression was detected.De Novo Software, Glendale, CA, USA).Caspase-1 activity and pyroptotic cell death by propidium iodine staining was measured in single cells using imaging Laser Scanning Cytometry (LSC). Mouse macrophages were cultured, treated, stained and imaged in 8 well IBIDI  slides with an initial concentration of 15,000 cells per well. Sub-vital staining was performed in culture medium at room temperature for 20 minutes by Hoechst 34580 (10 microg/ml), propidium iodine (10 microg/ml), Alexa 488 tagged Annexin V (1 microg/ml) and caspase-1 specific FLICA® 660 . In some experiments specific Caspase-1/ICE Inhibitor Z-WEHD-FMK  was also used before FLICA labeling. For LSC imaging an iCys Research Cytometer  was used with its iNovator Application Development Toolkit software. Hoechst, Alexa, PI and FLICA fluorescence dyes were excited separately with 405, 488, 488, 633 nm laser lines and detected at 430-470, 515-545, 650-700, 650 and above nanometers, respectively. Single cell data were gated according to their area, DNA content and nuclear shape and fluorescence pixel integral, maximum pixel intensity and average pixel intensity parameters with raw images were recorded for all dyes. For cytoplasmic caspase-1 activity measurements dynamic background subtraction was applied. Gated single cell FCS data were exported from LSC software and contour plots were generated in FCS Express 5 flow and image cytometry data analysis software (Heligmosomoides polygyrus life cycle was maintained in house and infective thirdstage larvae (L3) were obtained as described elsewhere  9 days after H.polygyrus infection. 6h after bacterial inoculation peritoneal macrophages were isolated by lavage, purified by adherence for 2 h to cell culture plastic and total RNA extracted.lsewhere . Mice wen SL3261  was cultn SL3261 . Brieflyex vivo. Cells were stained for surface markers then fixed with 2% paraformaldehyde (Sigma Aldrich), permeabilized using Permeabilization Buffer (eBioscience) and stained with directly labeled antibodies to NOS2  or biotinylated polyclonal goat antiYm1 (R&D Systems) followed by streptavidin-PerCP (Biolegend). Expression of Ym1 and NOS2 was determined relative to appropriate polyclonal or monoclonal isotype controls.All antibodies were purchased from Biolegend UK unless otherwise indicated. Equal numbers of cells were stained with LIVE/DEAD cell viability assay (Life Technologies) and blocked with 5 μg/mL anti CD16/32  and heatinactivated normal mouse serum (1:10) in FACS buffer (0.5% BSA and 2 mM EDTA in Dulbecco’s PBS) before surface staining with antibodies to F4/80 (BM8), SiglecF (E502440), Ly6C (HK1.4), Ly-6G (1A8), TCRβ (H57-597), CD11b (M1/70), CD11c (N418), I-A/I-E (M5/114.15.2), CD19 (6D5) and CD115 (AFS98). Detection of intracellular Ym1 and NOS2 was performed directly Samples were acquired on a BD LSR II using BD FACSDiva software (BD Bioscience) and post-acquisition analysis performed using FlowJo v9 software (Tree Star Inc.). Macrophages were identified as lineage negative , CD11b+ CD115+.Stat6−/− macrophages exposed to IL-4 for 1, 3, 6 and 24 hours in R using the aov and TukeyHSD functions of the MASS package. Differences were considered statistically significant at p value < 0.05 and FC > 2. For IL-4 pretreatment-LPS datasets, LPS-induced genes were considered statistically significant at p value < 0.05 compared to the control and then these genes were clustered based on their sensitivity  to IL-4 pretreatment as follows: attenuated response - Cluster 1; insensitive - Cluster 2; increased response - Cluster 3. K-means clustering was performed in R using the function kmeans from package stats. Gene Set Enrichment Analysis was done by GSEA v2.2.0  from HOMER. The HOMER option ‘-style groseq’ was used for GRO-seq samples. Integrative Genomics Viewer  was used for data browsing  samples were downloaded from the Sequence Read Archive of NCBI and processed using our ChIP-seq analysis command line pipeline . ConsensStatistical analysis for qRT-PCR, ChIP-qPCR, ELISA, LDH-release assay, FACS analysis and densitometry analysis of western blot: the error bars represent standard deviation (SD). The two-tailed Student’s t test was used to evaluate the significance of differences between two groups. Quantification and alignments of NGS analysis for RNA-seq, ChIP-seq and ATAC-seq are also described in more detail in the methods section above.GSE106706. Microarray and RNA-seq datasets were downloaded from NCBI GEO depository (GSE33609) and ArrayExpress (E-MTAB-995). ChIP-seq datasets were downloaded from NCBI GEO depository (GSE27060 and GSE38379) as well as Sequence Read Archive (SRP019970). The used genome-wide datasets are collected in The accession number for the RNA-seq, ChIP-seq, GRO-seq, and ATAC-seq data reported in this paper is GEO:


Drosophila. In wild-type photoreceptors, Syx5 localizes to cis-Golgi, along with cis-Golgi markers: Rab1 and GM130. We observed that Syx5-deficient photoreceptors show notable accumulation of these cis-Golgi markers accompanying drastic accumulation of vesicles between endoplasmic reticulum (ER) and Golgi cisternae. Extensive analysis of Rh1 (rhodopsin 1) trafficking revealed that in Syx5-deficient photoreceptors, Rh1 is exported from the ER with normal kinetics, retained in the cis-Golgi region along with GM130 for a prolonged period, and then subsequently degraded presumably by endoplasmic reticulum-associated protein degradation (ERAD) after retrieval to the ER. Unlike our previous report of Rab6-deficient photoreceptors – where two apical transport pathways are specifically inhibited – vesicle transport pathways to all plasma membrane domains are inhibited in Syx5-deficient photoreceptors, implying that Rab6 and Syx5 are acting in different steps of intra-Golgi transport. These results indicate that Syx5 is crucial for membrane protein transport, presumably during ER-derived vesicle fusion to form cis-Golgi cisternae.SNAREs (SNAP receptors) are the key components of protein complexes that drive membrane fusion. Here, we report the function of a SNARE, Syntaxin 5 (Syx5), in the development of photoreceptors in  Summary: Unlike Rab6-deficiency which affects only apical transport pathways, Syx5-deficiency inhibits all of polarized transport pathways, implying that these two genes are working in distinct stages of intra-Golgi transport. Drosophila photoreceptors, massive biosynthetic membrane traffic delivers rhodopsin to the rhabdomeres    . Therefore, we conclude that the phenotype of ER membrane amplification is not obvious in photoreceptors of late pupal stages.In a previous study, ectopic expression of ER-resident KDEL receptor in Syx5-deficient fat body cells displayed abnormal localization, suggesting ER membrane malformation . Thus, wcis-Golgi cisternae), and Rab1 (on COPII vesicles) was notably increased in Syx5-deficient photoreceptors  containing Rh1, indicating that Rh1 is degraded within MVBs, however, it takes more than 3 h to degrade Rh1 in the MVBs  method . Rh1 comthe MVBs . TherefoSyx5 mutant photoreceptors. ER degradation enhancer, mannosidase alpha-like 1(EDEM1) and 2 (EDEM2) were reported to be involved in the degradation of misfolded P23H mutant rod opsin protein by ERAD in vertebrate . ERAD not only eliminates terminally misfolded or unassembled polypeptides, but also degrades surplus polypeptides, thereby regulating the level of enzymes and other molecules . Therefortebrate . Therefoytoplasm B,C. TRP ytoplasm A,B. Howeeceptors C. The dieceptors . These rEP2313yx5, or EP2313 ex2Syx5 mosaic retinas and EP2313Syx5, or EP2313 ex2Syx5 whole-eye clones of newly eclosed flies under the electron microscope  MVBs per cross-section of a wild-type photoreceptor and 0.05 (s.d.±0.02) MVBs per cross-section of a EP2313Syx5 photoreceptor.In Syx5-deficient cells, late pupal stage ER appear healthy in contrast to the dilated ER in the photoreceptors expressing Rab1 dominant negative mutant protein . Consistild-type . Howeveri and ER D,E. ThesSyx5 mutants, we observed that EP2313 ex2Syx5 Golgi units contained more vesicles as compared to the wild-type Golgi units, though the overall Golgi structure in EP2313 ex2Syx5 mutant was normal  were absent.On comparing the Golgi units in wild-type and s normal F. To qualgi unit G. Considild-type , howevercis-Golgi in a normal time course but remains there for a prolonged period when compared to that in the wild-type cells. The accumulated Rh1 on cis-Golgi is then most likely degraded by ERAD. Using electron microscopy, we observed massive vesicle accumulation between ER and Golgi units in Syx5-deficient photoreceptors. Massive accumulation of the COPII tethering factors, Rab1, and GM130 was also observed in the Golgi units of Syx5 mutants. Therefore, we believe that in Syx5-deficient photoreceptors, the accumulated vesicles are most likely the COPII vesicles, thus, suggesting that Syx5 is involved in the fusion of COPII vesicles to cis-Golgi cisternae. These results are in agreement with the studies carried out in cultured cells  lines was crossed to EP2313, FRT40A/CyOSyx5, or EY07901, FRT40A/CyOSyx5 line. Then EP2313, FRT40A/CyO; ry, Sb, Delta2-3/+Syx5 males were crossed with the females of tester lines for live-imaging. The mosaic eye phenotypes of male progenies with the genotype, EP2313 ex, FRT40A/CyO; Rh1Arr2GFP eye-FLPSyx5  or EY07901 ex, FRT40A/CyO; Rh1Arr2GFP eye-FLPSyx5  were observed using confocal microscope. Some of them were crossed to Sco/Cyo and a couple of excision lines, EP2313 ex, FRT40A/CyOSyx5  or EY07901 ex FRT40A/CyOSyx5  were established. For the analysis of EP1588EDEM1, DG03809EDEM2  and EP2313Syx5  triple deficient photoreceptors: EP1588w, EDEM1 ; DG03809, Syx5 EP2313, FRT40AEDEM2  were crossed with EP1588; P3RFP FRT40A/SM1y w EDEM1  and ey-FLP/+.Flies were grown at 18–25°C on standard cornmeal-glucose-agar-yeast medium, unless indicated otherwise. For live-imaging and immunostaining in the Bruin flies, the following tester lines were used: staining . For P-eRh1-Gal4, GMR-Gal4, EGUF40A , EP1588EDEM1 , DG03809EDEM2   UAS-CFP::GalT .The following fly stocks were used: FP::GalT  and UAS-Fluorescent proteins expressed in the photoreceptors were imaged by a water-immersion technique as described previously .+K+ATPase alpha subunit (1: 500 ascites) , rabbit anti-TRP (1:1000) , Rabbit anti-Rab7 (1:1000) , mouse monoclonal anti-Eys (1:20 supernatant) , Rat monoclonal anti-DECad (1:20 supernatant) , mouse monoclonal anti-Chp (1:20 supernatant)  and rabbit anti-Chc (1:500) .The samples were fixed and stained as per the protocol described previously . PrimarySecondary antibodies used were: anti-mouse, -rabbit, -rat, and/or -chicken labeled with Alexa Fluor 488, 568, or 647 (1:300) . Samples were analyzed and images were recorded using a FV1000 confocal microscope .all-trans-retinal  in the dark. After one or two nights in the dark, flies were irradiated with a 405 nm diode laser module at 30 mW for 20 min  to isomerize the all-trans retinal to 11-cis-form and initiate Rh1 exit from the ER.Newly eclosed flies fed on carotenoid-deprived food  were switched to carotenoid-deprived food supplemented with crystalline Electron microscopy was performed as described previously . Samples


Type I collagen increased PDAC tumor initiating potential, self-renewal, and the frequency of CSCs through the activation of FAK. FAK overexpression increased tumor initiation, whereas a dominant negative FAK mutant or FAK kinase inhibitors reduced clonogenic PDAC growth in vitro and in vivo. Moreover, the FAK inhibitor VS-4718 extended the anti-tumor response to gemcitabine and nab-paclitaxel in patient-derived PDAC xenografts, and the loss of FAK expression limited metastatic dissemination of orthotopic xenografts. Type I collagen enhances PDAC CSCs, and both kinase-dependent and independent activities of FAK impact PDAC tumor initiation, self-renewal, and metastasis. The anti-tumor impact of FAK inhibitors in combination with standard chemotherapy support the clinical testing of this combination.Cancer stem cells (CSCs) play an important role in the clonogenic growth and metastasis of pancreatic ductal adenocarcinoma (PDAC). A hallmark of PDAC is the desmoplastic reaction, but the impact of the tumor microenvironment (TME) on CSCs is unknown. In order to better understand the mechanisms, we examined the impact of extracellular matrix (ECM) proteins on PDAC CSCs. We quantified the effect of ECM proteins, β1-integrin, and focal adhesion kinase (FAK) on clonogenic PDAC growth and migration  Pancreatic ductal adenocarcinoma (PDAC) leads to an estimated 40,000 deaths annually and is the fourth leading cause of cancer related deaths in the United States . DespiteFAK1) is a multifunctional non-receptor protein tyrosine kinase, encoded by the PTK2 (protein tyrosine kinase 2) gene located at chromosome 8q24.3. FAK is required for normal embryonic development ..54].in vivo PDAC growth, we injected shFAK-expressing cells into mice and initiated treatment with doxycycline following the development of tumors. Compared to control shRNA cells, FAK knock down led to a regression of tumors by the end of 3 weeks of treatment  from tumor associated macrophages , 61, 62.FAK is pleiotropic and in addition to its kinase activity, the FERM and FAT domains provide multiple docking sites for several cellular factors including growth factor receptors, ARP2/3, Endorphilin-A2, and the E3 ubiquitin-protein ligase MDM2 (Mouse double minute 2 homolog) . This scin vitro and in vivo. Pharmacologic FAK inhibitors have been developed and demonstrated to be active against PDAC in preclinical studies [18F-fluorodeoxyglucose positron emission tomography in three out of six patients with PDAC [The effects of type I collagen on PDAC CSCs is mediated through the activation of FAK. FAK is an intracellular, multifunctional protein tyrosine kinase that interacts with integrins at the plasma membrane in response to extracellular stimuli. Structurally, it has three distinct domains: an amino-terminal FERM (band 4.1–Ezrin–Radixin–Moesin) domain; a central kinase domain; and a C-terminal focal adhesion-targeting domain . Upon ac studies , 40. Furith PDAC . Similarith PDAC , we founBxPC-3, Capan-1 and MIA PaCa-2 cell lines were purchased from American Type Culture Collection and cultured on 60 mm dishes coated with or without type I collagen, fibronectin, or laminin (BD Biosciences). Prior to analysis of clonogenic growth or migration, cells were washed with 1X PBS, trypsinized, and re-suspended in medium containing 10% fetal bovine serum. Treatment with FAK-inhibitors PF573228 and VS-4718 was carried out for 72–120 hours on plastic or type I collagen.Low passage xenografts generated from surgical specimens derived from patients undergoing surgery at the Johns Hopkins Hospital have been previously described , 69. A tFull-length FAK (FAK-FL) was cloned into the NotI and BamHI sites of the pLVX-IRES-mCherry lentiviral vector (Clontech). Y397F-FAK was created using the QuikChange site-directed mutagenesis kit (Stratagene). Cell lines transduced with lentiviral constructs were selected by fluorescence activated cell sorting (FACS) based on mCherry expression. Short hairpin RNA (shRNA) constructs against FAK (shFAK-1 and shFAK-2) were cloned into the Tet-pLKO-puro lentiviral vector (Addgene). Cell lines were transduced and stable lines were selected using puromycin (Thermo Fisher Scientific).All experiments using mice were approved by the Johns Hopkins University Animal Care and Use Committee and the mice were maintained in accordance with the American Association of Laboratory Animal Care guidelines. In all studies, the appropriate measures were taken to minimize discomfort to animals. All injections and surgical procedures were performed using sterile technique with efforts made to minimize trauma to the animals. When necessary, animals were anesthetized with a mixture of 1.75% isofluorane/air. Following injections, animals were closely monitored and any that appeared moribund were immediately euthanized by administration of anesthesia, followed by inhalation of carbon dioxide until breathing ceased. Death was then ensured through cervical dislocation. Tumor growth was followed weekly by caliper measurements. For drug studies nude mice with subcutaneous low passage xenograft (JH102) were treated with VS-4718  and / or gemcitabine  and nab-paclitaxel . Tumors were measured as previously described , 69. TumTumor colony formation assay was carried out as previously described , 69ACTB  or ITGB1 (Hs00559595_m1).Total RNA was extracted using the RNeasy PlusMini Kit (Qiagen) and reverse-transcribed with SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was carried out using TaqMan probe against ® Novex® 4–12% Bis-Tris Protein Gels (Life technologies) and transferred to PVDF membrane. Membranes were blocked by 5% w/v BSA, 1X TBS and 0.1% Tween-20 at room temperature for one hour. Membranes were then incubated with rabbit monoclonal anti-pY397-FAK, rabbit polyclonal anti-FAK , beta-tubulin, or GAPDH (Abcam) antibodies. Densitometic analyses of protein expressions were done by ImageJ software.Whole cell lysates (WCL) from cells were prepared using pre-chilled Mammalian Protein Extraction Reagent (Life technologies) and incubated 30 minutes on ice. The lysates were cleared by centrifugation at 13,400 rpm and 4°C for 15 min. Immunoprecipitation was carried out by incubating 500 μg of WCL with 1–2 μg of anti-FAK antibody at 4°C for 16 hours. Subsequently, 25 μg of recombinant protein A agarose beads (Thermo Scientific) were added and incubated for one hour at 4°C with slight agitation. For Western blot, proteins were separated by electrophoresis using NuPAGE+ cells were identified by based on cells stained with Aldefluor in the presence of diethylaminobenzaldehyde (DEAB), a specific inhibitor of ALDH, used to control for background fluorescence. For intracellular staining, cells were fixed with 2% formalin for 10 minutes, followed by permeabilization with 1% NP40 for 10 minutes at room temperature. Cells were stained with rabbit monoclonal anti-pY397-FAK (Invitrogen), rabbit polyclonal anti-FAK , or mouse monoclonal anti-ALDH (BD Biosciences) antibodies followed by anti-rabbit-APC and anti-mouse-FITC antibodies (Invitrogen). Annexin V staining was done according to manufacturer’s protocol (eBioscience).Flow cytometry was carried out as previously described , 69. ForTumor-initiating cell (TIC) frequency was calculated by injecting 10, 100, 500, 1000 or 10,000 MIA PaCa-2 cells expressing control (vector) or FAK-FL or FAK-Y397F subcutaneously into NSG mice. TIC frequency was calculated using extreme limiting dilution analysis (ELDA) .Cell migration assay was carried out as previously described , 69. BriCells expressing inducible shRNA were treated with 50 ng/ml doxycycline for 96 hours and then RNA was isolated using RNeasy Mini Kit (Qiagen). Gene expression analysis was performed on an Illumina Human HT12_4 array at the Johns Hopkins University Sidney Kimmel Comprehensive Cancer Center Microarray Core Lab. Microarray data are available at Gene Expression Omnibus (GEO) under the accession no. GSE97488. Gene set enrichment analysis was performed with JAVA-based GSEA software .Statistical differences between two groups were analyzed using the two-tailed, unpaired Student t test or, if described, by ANOVA .S1 Figa) Relative mRNA and protein expression following knockdown of β1 integrin by shRNA in MIA PaCa-2 cells. (b) Primary and secondary colony formation by Capan-1 cells with scrambled control (shCtrl) or β1 integrin (shBeta1) shRNA after growth on type I collagen for 96 hours. Data represents mean ± SD (n = 4). **P < 0.001. (c) Cell viability of MIA PaCa-2 cells following knockdown of β1 integrin as assessed by annexin V staining.((TIF)Click here for additional data file.S2 FigPhospho-FAK (pFAK) and total FAK expression in MIA PaCa-2 and Capan-1 cells following overexpression of FAK-FL. Beta Tubulin was used as a loading control.(TIF)Click here for additional data file.S3 Figa) α2β1 integrin expression by ALDH+ or all MIA PaCa-2 cells. (b) Phospho-FAK expression by ALDH+ or all MIA PaCa-2, and Capan-1 cells. (c) Frequency of ALDH+ MIA PaCa-2 cells following overexpression of FAK-FL. Data represents mean ± SD (n = 3). (d) Frequency of ALDH+ MIA PaCa-2 cells expressing scrambled control (shCtrl) β1 integrin (shBeta1) shRNA. Data represents mean ± SD (n = 3). *P < 0.05.((TIF)Click here for additional data file.S4 Figa) In vitro colony formation by MIA PaCa-2 and Capan-1 cells following treatment with vehicle control (DMSO) or PF573228 on collagen I for 96 hours. Data represent mean ± SD (n = 4) of DMSO versus PF573228; **P < 0.001. (b) Colony formation by patient derived xenograft cells following treatment with vehicle control (DMSO) or PF573228 for 5 days. (c) Frequency of phopho-FAK+ (pFAK) and ALDH+ cells in Capan-1 and MIA PaCa-2 cell lines following treatment with DMSO or PF573228 (top panel) on type I collagen for 96 hours. Relative frequency of ALDH+ cells in patient derived xenografts following treatment with vehicle control (DMSO) or PF573228 (bottom panel). (d) Cell viability of MIA PaCa-2 cells following treatment with DMSO or VS-4718 as assessed by annexin V staining. (e) Ratio of pFAK to total FAK expression by JH102 xenograft cells following treatment with VS-4718, Gemcitabine plus nab-paclitaxel (Abraxane) and Gemcitabine plus nab-paclitaxel plus VS-4718. (f) Phospho-FAK expression in MIA PaCa-2 and Capan-1 cells overexpressing FAK-Y397F. Data represent ratio of phospho-FAK vs total FAK in percent (%).((TIF)Click here for additional data file.S5 Figa) FAK expression following knockdown by shRNA in MIA PaCa-2 cells. (b) In vitro cell proliferation by MIA PaCa-2 cells expressing shFAK following treatment with doxycycline for 3 days. (c) Cell viability of MIA PaCa-2 cells following knockdown of FAK as assessed by Annexin V staining.((TIF)Click here for additional data file.S6 FigP < 0.01 and FDR q = 0.01).GSEA analysis revealed that embryonic stem cell genes signature is significantly overlapped with gene expression data set of PDAC cells expressing FAK. Control / FAK hairpin data set versus Wong cancer stem cell core data set, Click here for additional data file.S7 Figa) In vitro migration by BxPC-3, Capan-1 and MIA PaCa-2 cells following growth on collagen I, fibronectin, or laminin for 96 hours. Data represent mean ± SD (n = 4) compared to control; *P < 0.05; **P < 0.001. (b) Migration by MIA PaCa-2 and Capan-1 cells expressing shBeta1 following growth on collagen I for 96 hours. Data represent mean ± SD (n = 4) of control versus shBeta1; *P < 0.05. (c) In vitro migration by MIA PaCa-2 cells following treatment with DMSO or VS-4718 on collagen I for 96 hours. Data represent mean ± SD (n = 4) of DMSO versus VS-4718; *P < 0.05. (d) Hematoxylin and eosin (H&E) staining of orthotopically grown MIA PaCa-2 tumors and metastases.((TIF)Click here for additional data file.