Regulation of capsid disassembly is crucial for efficient HIV-1 cDNA synthesis after entry, yet host factors involved in this process remain largely unknown. Here, we employ genetic screening of human T-cells to identify maternal embryonic leucine zipper kinase (MELK) as a host factor required for optimal uncoating of the HIV-1 core to promote viral cDNA synthesis. Depletion of MELK inhibited HIV-1 cDNA synthesis with a concomitant delay of capsid disassembly. MELK phosphorylated Ser-149 of the capsid in the multimerized HIV-1 core, and a mutant virus carrying a phosphorylation-mimetic amino-acid substitution of Ser-149 underwent premature capsid disassembly and earlier HIV-1 cDNA synthesis, and eventually failed to enter the nucleus. Moreover, a small-molecule MELK inhibitor reduced the efficiency of HIV-1 replication in peripheral blood mononuclear cells in a dose-dependent manner. These results reveal a previously unrecognized mechanism of HIV-1 capsid disassembly and implicate MELK as a potential target for anti-HIV therapy. Phosphorylation of the HIV-1 capsid has long been known to regulate viral uncoating and cDNA synthesis processes, but the cellular kinases responsible for this have remained unidentified. Here, we report that a host cell kinase MELK dictates optimal capsid disassembly through phosphorylation of Ser-149 in the multimerized HIV-1 core, which leads to efficient viral cDNA synthesis in target cells. The phosphorylation-mimetic capsid mutation of Ser-149 caused aberrant capsid disassembly and too-early completion of reverse transcription, and impeded nuclear entry of HIV-1 cDNA, suggesting the importance of well-ordered capsid disassembly in the early stages of viral replication. This discovery will facilitate understanding of the functional link among virus uncoating, reverse transcription and nuclear entry, and is expected to contribute to developing a novel strategy for AIDS therapy. During the course of human immunodeficiency virus type 1 (HIV-1) infection, the virus encounters numerous bottlenecks constituted by a variety of host cell proteins essential for or inhibitory to HIV-1 replication –5]. It h>75,000 shRNAs directed against >15,000 human genes in total. Transduced cells were then infected with the HIV-1NL4-3 strain which normally kills infected MT4C5 cells. Surviving cells were then studied further  library comprising  further . Using t further . Of thesMELK mRNA  . Lentivirus-mediated stable expression of a MELK-targeting shRNA, but not that of non-targeting shRNA (Non-T), suppressed expression of ELK mRNA . AnalyseELK mRNA . DepletiELK mRNA . ImportaELK mRNA . In contELK mRNA . Viral DWe first investigated whether MELK affects HIV-1 entry using the HIV-1 virion fusion assay with β-lactamase-Vpr chimeric protein incorporated into HIV-1 virions. This approach revealed similar efficiencies of HIV-1 entry into control Non-T and MELK-KD MT4C5 cells and a marked inhibition of HIV-1 fusion in the presence of the CXCR4 antagonist AMD3100  Fig 2A . The envin vitro luminescent kinase assay, in which the amount of ADP produced in the kinase reaction was quantified  of MELK (MT4C5-MELK-KD-1) with a lentivirus vector capable of expressing wild-type MELK substantially restored HIV-1 infectivity in two independent cell pools . To deteT-HIV-CA . In vitrt manner . These rWe next explored whether an amino-acid substitution that mimics phosphorylation of each serine or threonine residue, Thr-119, Ser-146, Thr-148 and Ser-149, counteracts the delay of CA disassembly and reduction in viral cDNA synthesis caused by MELK depletion. We generated four mutant pNL4-3 proviral molecular clones in which each Ser or Thr residue was substituted by a glutamic acid residue so that each mutation mimics constitutive phosphorylation of the site. Mutant HIV-1 bearing T119E, S146E, T148E, or S149E mutations in CA were used to evaluate the efficiency of viral cDNA synthesis in MELK-KD MT4C5 cells. The amount of each input CA-mutated virus was normalized by its RT activity. HIV-1 bearing T119E, S146E or T148E poorly restored early  and lateTo assess how the S149E mutation in CA influences the kinetics of capsid disassembly, we performed a fate-of-capsid assay in control Non-T and MELK-KD MT4C5 cells. The S149E substitution resulted in a clear decrease of the HIV-1 core not only in MELK-KD cells but also in Non-T cells, indicating that this mutation promoted the CA disassembly irrespective of the presence of MELK . SDS-PAGE   ablated CA phosphorylation LK in CA . The lacLK in CA . A previLK in CA . Our PhoLK in CA . This deIn conclusion, the essential role of MELK at an early stage of HIV-1 infection exemplifies another aspect of the functional links between viral capsid disassembly, cDNA synthesis and nuclear import. These findings contribute to our understanding of early viral life-cycle events and raise the possibility of developing a new class of anti-HIV agents targeting viral capsid disassembly.HEK293 , HEK293T , HeLa (ATCC) and TZM-bl [National Institutes of Health (NIH) AIDS Research and Reference Reagent Program] cells were propagated in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum (FBS) and penicillin/streptomycin. MT4C5  cells were maintained in complete RPMI 1640 medium supplemented with 10% FBS and penicillin/streptomycin. LuSIV cells (NIH AIDS Research and Reference Reagent Program) were cultured in RPMI 1640 supplemented with 10% FBS, penicillin/streptomycin, and 300 μg per ml hygromycin B. Phytohemaggulutinin (PHA)-activated PBMCs (PHA-PBMCs) were cultured in RPMI 1640 containing 10% FBS, penicillin/streptomycin, and 100 U IL-2 per ml. CD3/CD28-stimulated peripheral blood lymphocytes (PBL) were prepared using human T-Activator CD3/CD28 Dynabeads  and cultured in RPMI 1640 containing 10% FBS, penicillin/streptomycin, and 100 U IL-2 per ml.Nevirapine (NVP) and Azidothymidine (AZT) were obtained from National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. AMD3100 and MG132 were obtained from Sigma-Aldrich. Siomycin A was obtained from Bioaustralis. OTSSP167 was obtained from Selleck chemicals.env-/nef-) plus 2 μg of pHCMV-G (VSV-G) or pLET (HIV-1LAI/IIIB envelope), using FuGENE 6  as recommended by the manufacturer. Virus stocks used for analysis of viral replication were prepared by transfection of HeLa cells using LipofectAMINE LTX PLUS  with molecular clone DNAs of HIV-1NL4-3, pNL4-3  and incubated on ice for 15 min. Swollen cells were lysed in a 7 ml-Dounce homogenizer with a ‘tight’ pestle  and cell lysates cleared by centrifugation at 2,000 × g for 3 min at 4°C. Cleared cell extracts (0.8 ml) were layered over 20%–60% sucrose cushions prepared in PBS and centrifuged at 4°C and 35,000 rpm for 70 min in a Beckman SW50.1 rotor; 50 μl of the cell extract was reserved as a ‘cell lysate’ fraction. After centrifugation, three fractions of 1.1 ml each were collected from the top of the gradient. Aliquots of each fraction of the step gradients were subsequently processed for immunoblotting. The amount of CA protein in each fraction was quantified using HIV-1 CA (p24) enzyme-linked immunosorbent assay kits .The fate-of-capsid assay was performed with minor modifications  as previEnvelope-stripped HIV-1 cores were prepared as described previously . BrieflyEnvelope-stripped HIV-1 cores isolated by ultracentrifugation were absorbed onto Formvar-coated copper grids, and stained with 2% phosphotungstic acid solution. The images were recorded with a Tecnai F20 transmission electron microscope  at 200kV.HeLa cells were transfected with pCAG-OSF-MELK, pCAG-OSF-GFP, pCAG-OSF-CypA, or pCAG-FOS2-rhT5α, harvested 48 h post-transfection and lysed in a 7 ml-Dounce homogenizer. Cell extracts were incubated with Strep-Tactin Sepharose for 2 h at 4°C. Purified Strep-tagged protein complexes were incubated with envelope-stripped HIV-1 cores  for 2 h at 4°C. After extensive washing, Strep-tagged protein complexes were released by boiling in SDS-PAGE loading buffer and the proteins were analyzed by 12% SDS-PAGE and Western blotting using mouse anti-FLAG antibody (FLAG) and mouse anti-p24 antibody (CA).E.coli BL21 CodonPlus-RIL cells  transformed with pGEX-4T-3 or pGEX-HIV-CA were used for purification of GST proteins using standard methods. Fifteen independent synthetic peptides covering HIV-1 CA were designed and provided by Sigma-Aldrich . The amount of CA protein was quantified using HIV-1 CA (p24) enzyme-linked immunosorbent assay kits .In vitro phosphorylation assays were performed with the ADP-Glo MELK kinase assay kit, following the manufacturer’s instructions . Briefly, 100 ng of recombinant activated MELK was incubated with either GST, GST-HIV-CA or Env-stripped HIV-1 core proteins ranging from 100 to 2,000 ng or synthetic peptides, ZIPtide, ranging from 500 to 2,000 ng for 60 min at 30°C in the presence of ultrapure ATP. Light emission was measured using the GloMAX multidetection system . ZIPtide was used as a phosphorylation standard for MELK. For the detection of phosphorylated CA protein in the multimerized viral core, 200 ng of recombinant activated MELK was incubated with envelope-stripped HIV-1 core containing 100 ng of p24 at 30°C in the presence of ultrapure ATP. The phosphorylation reaction was terminated by the addition of 2 × sample buffer. The proteins were subsequently processed for immunoblotting using rabbit polyclonal antibodies to phospho-S149-CA, mouse monoclonal antibody to HIV-1 p24 , mouse monoclonal antibody to HIV-1 p17 , goat polyclonal antibody to gp120 , or rabbit monoclonal antibody to MELK .6 Non-T or MELK-KD MT4C5 cells were inoculated together with 2 × 107 RT counts of wild-type HIV-1 (HIV-1NL4-3 strain) or 5 × 106 RT counts of VSV-G/NL4-3luc or VSV-G/NL4-3 CA-S149A viruses. After incubation at 4°C for 30 min, cells were further incubated at 37°C for 8 h. Cells were then washed twice with ice-cold PBS(-) containing 0.005% Trypsin/EDTA to remove virions from the cell surface and once with ice-cold PBS(-) to remove Trypsin/EDTA. Washed cells were resuspended in 1 ml of hypotonic lysis buffer  and incubated on ice for 15 min. Swollen cells were lysed in a 7 ml-Dounce homogenizer with a ‘tight’ pestle  and cell lysates cleared by centrifugation at 2,000 × g for 3 min at 4°C. Proteins were separated in 10% precast SDS-polyacrylamide gels prepared with 50 μM acrylamide-pendant Phos-tag ligand  and were analyzed by immunoblotting with mouse anti-p24 antibody.Briefly, 5 × 10t-test. All the statistical analyses were performed using Prism 6 software . P values below 0.05  were considered significant. Unpaired two-tailed Student’s t-test was used for the data shown in All data are obtained from at least three independent experiments. The average values are presented with error bars indicating the standard deviation (SD) and the statistical significance was analyzed using one-way analysis of variance (ANOVA) with Dunnett’s or Tukey’s multiple comparison tests, two-way analysis of variance (ANOVA) with Tukey’s or Sidak’s multiple comparison test, or Student’s S1 Methods(DOCX)Click here for additional data file.S1 TableNo: Number of identified host factor in this screen; Symbol; gene symbol; 21 mer target sequence: shRNA sequences identified in infection-resistant cells free from HIV-1 DNA; GPP Clone ID: Identification data of shRNA provided by Genetic Perturbation Platform (GPP); GO-Biological Process: Identified biological process based upon gene ontology; GO-Cellular component: Identified cellular component based upon gene ontology. Information on gene ontology shown in this table was obtained from the Gene Ontology Annotation Database (http://www.ebi.ac.uk/GOA).Explanation of column headings in the table: (XLSX)Click here for additional data file.S1 FigCells were stained with anti-CD4 (left panels) or anti-CXCR4 mAb (right panels). As controls, MT4C5 cells were stained with isotype control mAbs (top panels).(TIFF)Click here for additional data file.S2 Fig0/G1, G0/G1, S and G2/M) are shown . Error bars are standard deviations calculated from five independent experiments. Statistical significance was determined by unpaired two-tailed Student’s t test. ns, not significant (P>0.05).Effects of MELK depletion on cell cycle progression were determined using propidium iodide (PI) staining and FACS analysis. Non-T and MELK-KD-2 MT4C5 cells were treated with  or without  0.05 μg/ml Demecolcine  for 16 h to synchronize them in M phase. Demecolcine was then removed and the cells were cultured for 24 h with fresh growth medium, stained with PI, and analyzed by FACS . Graphs show the distribution of cells in distinct cell cycle phases from five independent experiments (bottom panels). The average percentages of cells in each cell cycle phase (SubG(TIFF)Click here for additional data file.S3 Fig(A) Lysates of HEK293 cells stably expressing non-target shRNA or MELK-specific shRNA  were immunoblotted with anti-MELK or anti-alpha-tubulin antibodies. (B) Total RNA from the cells in (A) was extracted and examined for MELK mRNA expression by multiplex RT-PCR amplification (MELK). The primer set for amplification of GAPDH mRNA was included in each reaction as an internal control (GAPDH). (C) Effect of MELK depletion on single-round HIV-1 infection in HEK293 cells. HEK293 cells described in (A) and (B) were infected with VSV-G-pseudotyped NL4-3luc. The mean luciferase value from non-target shRNA HEK293 cells was arbitrarily set as 100%. Error bars reflect the standard deviations calculated from five independent experiments. (D) Lysates of CD3/CD28-stimulated PBLs stably expressing non-target shRNA or MELK-specific shRNA (PBL-MELK-KD-2 and 3) were immunoblotted with anti-MELK or anti-alpha-tubulin antibodies. (E) Total RNA was extracted and MELK mRNA expression determined by multiplex RT-PCR amplification (MELK). A primer set for amplification of GAPDH mRNA was included in each reaction as an internal control (GAPDH). (F) Effect of MELK depletion on a single-round of HIV-1 infection in CD3/CD28-stimulated PBL. PBL, Non-T, PBL-MELK-KD-2 and PBL-MELK-KD-3 cells described in (D) and (E) were infected with VSV-G-pseudotyped NL4-3luc. The mean luciferase value from non-target shRNA CD3/CD28-stimulated PBL was arbitrarily set as 100%. Error bars are standard deviations calculated from five independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (C and F). ns, not significant (P>0.05); *P<0.05, **P<0.01, ***P<0.001.(TIFF)Click here for additional data file.S4 Fig(A) HeLa cells were transfected with pCAG-OSF or pCAG-OSF-MELK. Transfected HeLa cell extracts were incubated with Strep-Tactin Sepharose and OSF-tagged proteins were purified. OSF-tagged control (lanes 1 and 3) and MELK (lanes 2 and 4) proteins were then incubated with purified HIV-1 virions (lanes 1 and 2) or envelope-stripped cores (lanes 3 and 4), and complex formation was assessed by immunoblotting (IB) using rabbit anti-MELK antibody (MELK) and mouse anti-p24 antibody (CA). (B) Purified OSF-tagged control (lane 1) and MELK (lane 2) proteins were incubated with soluble CA (input). Complex formation was assessed by immunoblotting (IB) using rabbit anti-MELK antibody (MELK) and mouse anti-p24 antibody (CA).(TIFF)Click here for additional data file.S5 FigEffect of MELK depletion on the fate of the HIV-1 CA in MT4C5 cells at 4 and 8 h post-infection analyzed as in (TIFF)Click here for additional data file.S6 Fig(A) Virion-associated viral RNA was quantified by quantitative RT-PCR 2 h after infection of Non-T or 293-MELK-KD-3 cells with wild-type HIV-1. (B) Effect of MELK depletion on the fate of the HIV-1 CA in HEK293 cells analyzed as in (C) Percentage of the pelletable CA within total CA as quantified by p24 ELISA shown in S6B Fig. (D) Non-T or MELK-KD-3 HEK293 cells were infected with VSV-G-pseudotyped HIV-1 for 2, 4 or 8 h in the presence or absence of the proteasome inhibitor MG132 . Whole cell lysates were immunoblotted with anti-p24 (CA) or anti-alpha-tubulin (α-tubulin) antibodies (left panels). Experiments were performed five times and one representative set of data is shown. The amounts of CA in the whole cell lysates were quantified by HIV-1 p24 ELISA (right panels). Error bars indicate the standard deviations calculated from five independent experiments. (E) Results of p24 ELISA showing the steady-state levels of CA in fraction #3 in and S6B Fig at the indicated time points . Statistical significance was determined by two-way analysis of variance (ANOVA) with Sidak’s multiple comparison test (D), or unpaired two-tailed Student’s t test . ns, not significant (P>0.05); *P<0.05, **P<0.01, ***P<0.001.(TIFF)Click here for additional data file.S7 FigPanel protein: Parental MT4C5 (lane 1), Non-T (lane 2) or MT4C5-MELK-KD-1 cells (lanes 3) transduced with control vector (lane 4) or vector for wild-type MELK  or catalytically inactive T167A MELK mutant  were used. Cell lysates were immunoblotted with anti-MELK or anti-α-tubulin antibodies. Panel mRNA: Total RNA from cells listed above was extracted. Total MELK mRNAs (upper panel), endogenous MELK mRNA (middle panel) and exogenous mutant MELK mRNA (bottom panel) were quantified by RT-PCR amplification with specific primer sets (MELK). The primer set for amplification of GAPDH mRNA was included in each reaction as an internal control (GAPDH). Experiments were performed three times and one set of representative data is shown.(TIF)Click here for additional data file.S8 FigPhosphorylation of recombinant CA by MELK was monitored as in (TIFF)Click here for additional data file.S9 Fig(A-F) Total DNA was extracted from non-target shRNA (Non-T) or MELK-depleted (MELK-KD-2) MT4C5 cells at the indicated time points  after wild-type or indicated mutants of HIV-1 infection and analyzed for the amounts of late RT product containing the env region. Experiments were performed at least three times and error bars are standard deviations calculated from three independent experiments. The ratios of each viral cDNA level to beta-globin DNA level are given. (G) Quantitative RT-PCR analyses of virion-associated viral RNA at 2 h after infection of Non-T or MELK-KD-2 MT4C5 cells with wild-type HIV-1 or CA S149E HIV-1 mutant. Error bars indicate the standard deviations calculated from five independent experiments. Statistical significance was determined by unpaired two-tailed Student’s t test (G). ns, not significant (P>0.05); *P<0.05, **P<0.01, ***P<0.001.(TIFF)Click here for additional data file.S10 Fig Virus production was monitored by assessing RT activity (A) or p24 antigen (B) in culture supernatants of HeLa cells. (C) Viral infectivity was evaluated by infection of TZM-bl (upper panel) and LuSIV (lower panel) indicator cell lines with culture supernatants shown in (A) normalized by RT activity. Relative luciferase activities are shown as percentages (%) of that of NL4-3wt with standard deviations calculated from five independent experiments.(TIFF)Click here for additional data file.S11 Fig(A) MT4C5, Non-T and MELK-KD-2 cells were infected with VSV-G-env-pseudotyped NL4-3luc CA-wt or CA-S149A normalized by reverse transcriptase (RT) counts corresponding to 10 ng (p24) of VSV-G/NL4-3luc CA-wt. Relative luciferase activities are shown with standard deviations calculated from five independent experiments. (B) The CA-S149A results in each cell pool were compared on the same Y axis setting. Statistical significance was determined by unpaired two-tailed Student’s t test (A), or one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (B). ns, not significant (P>0.05); *P<0.05, **P<0.01, ***P<0.001.(TIFF)Click here for additional data file.S12 FigThe sequences are aligned with the HIV.HXB2 sequence. The arrow indicates the 149th amino acid from the N-terminus of HIV-1 capsid. Dashes indicate amino acid sequence identity.(TIFF)Click here for additional data file.S13 Fig(A) Immunoblot analyses with anti-MELK (upper panel) or α-tubulin (lower panel) monitoring endogenous MELK expression in MT4C5 cells treated with increasing amounts of Siomycin A. (B) Semi-quantitative RT-PCR analysis of MELK and GAPDH mRNA expression in MT4C5 cells described in (A). (C) Effect of Siomycin A on HIV-1 replication in MT4C5 cells. The virion-associated RT activity was monitored at the indicated time points in culture supernatants of MT4C5 cells treated with Siomycin A  and those of MELK-KD-2 (closed diamonds). Error bars reflect the standard deviations calculated from three independent experiments.(TIFF)Click here for additional data file.


Oncogene combination m-Ras/m-p53/m-PIK3CA efficiently transformed both K5+/K19- and K5+/K19+ cell lines in-vitro, as assessed by anchorage-independent soft agar colony formation assay. Significantly, while this oncogene combination induced a complete epithelial-to-mesenchymal transition (EMT) in K5+/K19- cell line, mostly epithelial phenotype with minor EMT component was seen in K5+/K19+ cell line. However, both K5+/K19- and K5+/K19+ transformed cells exhibited increased invasion and migration abilities. Analyses of CD44 and CD24 expression showed both cell lines had tumor-initiating CD44+/CD24low cell population, however transformed K5+/K19- cells had more proportion of these cells. Significantly, both cell types exhibited in-vivo tumorigenesis, and maintained their EMT and epithelial nature in-vivo in mice tumors. Notably, while both cell types exhibited increase in tumor-initiating cell population, differential EMT phenotype was observed in these cell lines. These results suggest that EMT is a cell type dependent phenomenon and does not dictate oncogenesis.Breast cancer is characterized into different molecular subtypes, and each subtype is characterized by differential gene expression that are associated with distinct survival outcomes in patients. PIK3CA mutations are commonly associated with most breast cancer subtypes. More recently PIK3CA mutations have been shown to induce tumor heterogeneity and are associated with activation of EGFR-signaling and reduced relapse free survival in basal subtype of breast cancer. Thus, understanding what determines PIK3CA induced heterogeneity and oncogenesis, is an important area of investigation. In this study, we assessed the effect of mutant PIK3CA together with mutant Ras plus mutant p53 on oncogenic behavior of two distinct stem/progenitor breast cell lines, designated as K5+/K19- and K5+/K19+. Constructs were ectopically overexpressed in K5+/K19- and K5+/K19+ stem/progenitor cells, followed by various  Comprin-vitro , 9. Morein-vitro . Furtherin-vitro . Therefoin-vivo and showed variations in incidence and latency for tumor and metastasis formation. K5+/K19- cells transformed by oncogene combination mRas/mp53/wtErbB2 generated primary tumors with shorter latency in comparison to K5+/K19- cells transformed by mRas/mp5/wtEGFR. Although, primary tumor onset was significantly delayed for mRas/mp5/wtEGFR transformed K5+/K19- cells, these cell lines exhibited similar latency for developing lung metastasis as that of K5+/K19- cells transformed by mRas/mp53/wtErbB2. We also observed that transformed K5+/K19+ cell type overall had a higher metastasis formation ability than transformed K5+/K19- cells [We previously demonstrated that overexpression of oncogene combinations mRas/mp53/wtErbB2 or mRas/mp53/wtEGFR efficiently transformed two different basal subtypes of mammary stem/progenitor cell lines  designated as K5+/K19- and K5+/K19+ . Both th9- cells . Given, in-vitro, increased levels of CD44+/CD24low tumor-initiating population; and in-vivo tumors when orthotopically implanted into mammary glands of NOD/SCID gamma (NSG) mice. Significantly, however only K5+/K19- cells showed a clear EMT phenotype both in-vitro and in-vivo, while K5+/K19+ cells displayed mostly epithelial phenotype with minor EMT. Taken together, this study suggest EMT is cell type dependent rather than oncogene dependent and EMT does not necessarily correlate with in vitro or in vivo oncogenic behavior of cells.We report that overexpression of oncogene combination mRas/mp53/mPIK3CA in both cell types induces complete transformation, as assessed by increased anchorage independence and increased invasion/migration Mutant p53-R249S in pLENTI-6 (purchased from Addgene) along with Invitrogen packaging vector  were transfected into 293FT packaging cells. Lentiviral supernatants were collected after overnight incubation in fresh DMEM media. TSA54 packaging cells were transfected with retroviral constructs, mutant H-Ras Q61L in pBABE-hygro or mPIK3CA-H1047R in pMSCV-puro vector, together with PIK plasmid for packaging, and viral supernatants were collected . K5+/K19- and K5+/K19+ stem/progenitor cell lines described previously , were inThe following antibodies were used for western blotting, immunofluorescence, flow-cytometry and IHC: mouse anti-human p53 (DO-1) (sc-126), mouse anti-human α-smooth muscle actin (SMA) (sc-32251), mouse anti-human vimentin (sc-6260)—all were from Santa Cruz Biotechnology. Mouse anti-human Ras (610001), mouse anti-human MUC1 (550486), FITC conjugated anti-CD24 (555427), PE-conjugated anti-CD44 (555479) and Alexa-488 conjugated E-Cadherin (560061), all were from BD Bioscience. Rabbit anti-human vimentin  was from Thermo Scientific. Rabbit anti-human K5 (PRB-160P) was from Covance. Rabbit anti-human PI3 Kinase p110 α (4255S) was from Cell Signaling.As described previously , briefly1 million cells were incubated with FITC conjugated anti-CD24 and PE-conjugated anti-CD44 at 4°C for 45 min. Following incubation, the cells were washed three times with washing buffer  and were then subjected to fluorescence-activated cell sorter (FACS) analysis.20,000 cells were plated in trans-well chambers (BD Biosciences) and incubated at 37°C for 13 or 23 hrs. Cells in upper chamber were cleared using cotton swab, while the cells in the bottom chambers were fixed with HEMA fixative (#122-911A), followed by staining with HEMA solution I (#122-911B) and HEMA solution II (#122-911C) for 5 min. each. Stained cells were counted under the inverted microscope.Protocol used for matrigel assay has been described previously , 17. BriProtocol was similar as described earlier (12). Briefly 40,000 cells were plated in ultra-low attachment 6 well plates (Corning) in mammary epithelial growth medium (MEGM), as described previously by Dontu et al. Each cell line had 6 replicates. Cells were fed with fresh medium on alternate days. Tumorspheres were counted under the microscope after 3 weeks of plating. Tumorpheres were trypsinized and plated again for secondary or tertiarytumorsphere assays.2 inhalation followed by cervical dislocation. Tumors were excised, fixed with 10% neutral buffered formalin, processed to prepare paraffin-embedded tumor blocks that were then sectioned for IHC. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of University of Nebraska Medical Center, Omaha, NE (Protocol No. 10-086-12-FC).6–8 weeks old immuno-deficient NSG mice (purchased from Jackson laboratories) were orthotopically injected with 1 million cells in DFCI-1 medium mixed with matrigel in 1:1 proportion  in the fAs described previously , 19, 4 μXenograft tumors were excised from NSG mice and then minced with sterilized scalpel in clean P-60 petri dishes. Minced tumors were cultured in either α-MEM or DFCI-1 medium for 1–2 weeks, following which the cells were trypsinized, and subjected to antibiotic  selection to isolate human cells.in-vitro transformation ability of K5+/K19- and K5+/K19+ transfectants, we performed anchorage independent growth assay. We observed that K5+/K19- and K5+/K19+ transfectants with oncogene combination mp53/mPIK3CA were able to form few small colonies in soft agar than their respective controls (in-vitro transforming abilities of both cell lines and as observed previously (12) transformed K5+/K19+ cells formed more colonies than transformed K5+/K19- cells  showed loose EMT-like colonies, while transformed K5+/K19+ (clone 21) showed epithelial phenotype . These ein-vitro also contribute to oncogenic behavior of cells in-vivo. Therefore, we first assessed migration and invasion abilities of the transformed cell lines in-vitro. Notably, transformed K5+/K19- as well as K5+/K19+ cells showed increased migration as well as invasion when compared to vector cells   . We also9+ cells .These rein-vitro as a result of mRas/mp53/mPIK3CA over-expression in K5+/K19- and K5+/K19+ cells is also observed in in-vivo setting, we implanted the transformed cell lines in mammary gland of NSG mice. The vector cells, as expected from our previous published results did not show any tumor formation [in-vivo setting. EMT like transformed K5+/K19- cells were more potent in forming tumors and all of the 4 mice formed primary tumors within 4 weeks of injection. Notably, transformed K5+/K19+ cells had a latency period slightly higher than transformed K5+/K19- cells where tumors were observed by 6th week of injection (data not shown).To confirm if the transformation ability observed ormation . Signifiin-vitro cultures of these tumors, as they displayed α-SMA/Vimentin+/MUC1- EMT-like phenotype (+/Vimentin+/MUC1+) with slight EMT phenotype  in both orthotopic tumor as well as in tumor derived cell lines. Tumors from both cell types showed expected expression of transfected genes  and mutant p53 in two different stem/progenitor cell types K5+/K19- or K5+/K19+ and then assessed the effect of the oncogene combination in driving pathogenesis from the two isogenic cell lines. We observed that mRas/mp53/mPIK3CA combination was extremely potent in driving oncogenic phenotype in-vitro  , 21, 30.We demonstrate that ectopic over-expression of m-Ras/m-p53/mPIK3CA combination into K5+/K19- and K5+/K19+ stem/progenitor cells while transformed both cell types, only induced EMT in K5+/K19- cells, reflecting differential intrinsic susceptibilities of K19- or K19+ cellular precursors for EMT.S1 Fig+/K19- and K5+/K19+ cells with vector or mp53/mPIK3CA oncogene combination, as assessed by anchorage independent growth assay. B) Quantification of colonies formed by different cells. Mean ± S.D. of a representative experiment done in triplicate is shown.A). Representative images (magnification 4X) of colonies from K5(TIF)Click here for additional data file.S2 Fig+/K19- (clone 62) or K5+/K19+ (clone 21) cells (magnification 10X). (B) Parental or transformed K5+/K19- or K5+/K19+ cells were analyzed by western blotting for the expression of EMT markers- E-Cadherin or Vimentin. β-actin was used as a loading control.(A) Phase contrast images showing differences in EMT phenotype in transformed K5(TIF)Click here for additional data file.S3 Fig+/K19- and K5+/K19+ cells in different passages. Indicated cell lines were cultured in low-attachment plates in MEGM media for 3 weeks. Spheres ≥200μm were quantified.A, B and C Quantification of tumorspheres formed by vector or mp53/mRas/mPIK3CA gene combination over-expressing K5(TIF)Click here for additional data file.


Lysine acetylation has emerged as a dominant post-translational modification (PTM) regulating tau proteins in Alzheimer’s disease (AD) and related tauopathies. Mass spectrometry studies indicate that tau acetylation sites cluster within the microtubule-binding region (MTBR), a region that is highly conserved among tau, MAP2, and MAP4 family members, implying that acetylation could represent a conserved regulatory mechanism for MAPs beyond tau. Here, we combined mass spectrometry, biochemical assays, and cell-based approaches to demonstrate that the tau family members MAP2 and MAP4 are also subject to reversible acetylation. We identify a cluster of lysines in the MAP2 and MAP4 MTBR that undergo CBP-catalyzed acetylation, many of which are conserved in tau. Similar to tau, MAP2 acetylation can occur in a cysteine-dependent auto-regulatory manner in the presence of acetyl-CoA. Furthermore, tubulin reduced MAP2 acetylation, suggesting tubulin binding dictates MAP acetylation status. Taken together, these results uncover a striking conservation of MAP2/Tau family post-translational modifications that could expand our understanding of the dynamic mechanisms regulating microtubules. The cell lysates were heated to 100°C for 10 min, rapidly cooled on ice for 20 min and centrifuged at 70,000xg for 30 min. Supernatants were dialyzed into FPLC buffer A , applied onto a HiTrap Sepharose HP IEX cation-exchange column , and eluted with a 0–0.4 M NaCl gradient using an AKTA-Pure FPLC system . The fractions were checked for the presence of the tau proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) followed by Coomassie Blue R-250 staining. The fractions containing enriched tau or MAP2c were pooled together and dialyzed against 100 mM sodium acetate buffer, pH 7.0. Importantly, all purified proteins were subject to a critical heat-denaturation step at 100°C to inactivate any endogenous bacterial enzymes and simultaneously enrich for heat-stable tau proteins. Proteins were concentrated using Amicon Ultra centrifugal filter units , and protein concentrations were determined using the bicinchoninic acid protein assay  with bovine serum albumin as the protein standard.Tau-K18 (2N4R-tau spanning residues 244–372), MAP2c fragments (spanning residues 280–398 of the full-length 467 residue rat MAP2c), and MAP4 fragments (spanning residues 925–1102 of full-length 1152 residue human MAP4) were purified using an AKTA-Pure FPLC chromatography system (GE). The proteins were expressed in Escherichia coli BL21(DE3) RIL strain. Bacteria were grown in LB media containing ampicillin at 37°C and induced with isopropyl-D -thiogalactopyranoside at a final concentration of 0.8 mM when the OD reached 0.6. After agitation for 2 h, cells were harvested by centrifugation. The pellet was re-suspended in high-salt RAB buffer -labeled acetyl-CoA (Perkin Elmer) was substituted for unlabeled acetyl-CoA. Reaction products were analyzed by resolving proteins on 15% SDS-PAGE gels followed by transfer to nitrocellulose membranes and phosphorimaging analysis using Storm software, or immunoblotting with the indicated antibodies. MAP-rich fractions containing bovine brain-derived MAPs were purchased from Cytoskeleton and desalted, where indicated, using Zeba Spin Desalting Columns (Thermofisher) prior to acetylation reactions. For tubulin competition experiments, purified brain tubulin (Cytoskeleton) was titrated into acetylation reactions at final concentration of 1–10 μM and preincubated for 10 min at 37°C prior to initiation of acetylation by addition of acetyl-CoA. All reactions were performed with a minimum of N = 3 independent experimental replicates.Tau, MAP2c, or MAP4 fragments (5 μM) were either mock acetylated with coenzyme A alone or modified 3H]-acetyl-CoA  at concentrations ranging from 2.5 μM to 1 mM acetyl-CoA, incubated at 37° C, and allowed to proceed for 1 hr. Reactions were stopped by the addition of 10 μl of 3 mM CoA, and 25 μl of the reaction was then spotted to a P81 filter paper (Whatman), washed in 10 mM HEPES pH 7.5 three times, and dried using acetone. Incorporated [3H]-acetyl-CoA was then measured using the Perkin Elmer Tri-Carb 2800TR Liquid Scintillation Analyzer, and the molar amount of acetyl groups incorporated were calculated from a standard curve of the radiolabeled acetyl-CoA.For filter-binding assays, reactions containing tau-K18, MAP2c, or MAP4 fragments were initiated by the addition of . Al. Al20]. QIVYK311 .in vitro and cell-based assays  in which either 2 or 3 lysines were acetylated in both MAP2c and MAP4. Further acetylation site comparisons identified a more general consensus motif [V-Q/K-I-X-X-X/K-K] present at the N-terminus of MAP2c repeats 3/4 and MAP4 repeats 2/3/4, in which several acetylated lysines were enriched. Consistent with these two regions acting as putative MAP acetylation motifs, tau proteins are similarly acetylated in repeat 1 at residue K259 [254K-N-V-K-S-K259] [275V-Q-I-I-N-K-K281] [306V-Q-I-V-Y-K311) [Acetylated lysine-containing peptides were identified for both MAP2c  and MAP4-K-K281] , and repin trans by CBP/p300 [in vitro acetylation reactions with radiolabeled [14C]-acetyl-CoA. Tau and MAP2c acetylation reactions were separated by SDS-PAGE gel, transferred to nitrocellulose membranes, and analyzed by autoradiography and phosphorimaging. In agreement with our previous study -acetyl groups onto MAP2c and MAP4 lysine residues was measured by quantification of radiolabeled fragments using a filter-binding assay that traps low-molecular weight MAP fragments but not free unconjugated [3H]-acetyl-CoA. MAP2c and MAP4 fragments that span the MTBR showed significant acetylation, which increased linearly as a function of increasing concentration of acetyl-CoA from 2.5–800 μM  abrogated acetylation, strongly supporting cysteine-mediated auto-acetylation -acetyl-CoA labeling . Using a pan-acetyl-lysine antibody, we observed an increase in acetylated MTs (~ 55 kDa), which correlated with an apparent reduction of MAP2 acetylation-immunoreactive bands (~ 70 kDa) in mature neurons from 11–22 DIV  that is incapable of acetylating MAP lysines . Active 1–22 DIV . Taken tHere, we provide the first evidence for conserved lysine acetylation as a mechanism regulating MAP2/Tau family members. Although acetylation has traditionally been viewed in the context of histones and gene transcription, recent proteomics approaches have identified acetylated targets in the cytoplasm and other organelles . For exain vitro acetylation sites, most of which cluster within the tau MTBR [in vivo using wild-type mouse brain [Our previous mass spectrometry analysis of either recombinant tau proteins or purified tau isolated from cultured cells identified four major acetylation sites in cells  and several additional se brain . Since tse brain . Using aK-N-V-K/R-S-K] and [V-Q/K-I-X-X-X/K-K]. Consistent with enhanced acetylation at lysine-rich motifs, recent studies indicate that lysine acetylation preferentially occurs in a general lysine-rich local environment, in which neighboring lysines facilitate primary lysine acetylation  consensus acetylation motif. High resolution NMR analysis indicates that the beginning of the tau repeats represent strong binding sites for microtubules [K-I-X-X-X/K-K] motifs.It is currently unclear why MAPs are redundantly acetylated at the N-terminus of repeats 2–4 within the general [V-Q/otubules , 32, sugotubules . In factotubules . Thus, Motubules . Based ond repeat, in particular, make an unusually large contribution to MT-binding that is abrogated upon neutralization via lysine acetylation [332VQIVTKK338), the 2nd repeat of MAP4 (992VQIVSKK998) and the 3rd repeat of MAP4 (1023VQIQNKK1029) are all targeted by acetylation, either singly or doubly, in an analogous manner  as a critical factor in disease pathogenesis that may distinguish tau from its close relatives, which could factor into the selective tau toxicity observed in AD and related tauopathies.S1 FigGGQVQIVTKKIDLSHVTSK (K337/K338), with significant ion scores and p-values Click here for additional data file.S2 Figin vitro (DIV). Neuronal lysates were harvested and analyzed by immunoblotting using pan-acetyl-lysine, MAP2, tubulin, and GAPDH antibodies. Acetyl-lysine immunoreactive protein bands correlated with MAP2 migrating bands at ~ 70 kDa (top arrow), just above the prominent ~ 55 kDa acetyl-lysine immunoreactive band that corresponds to acetylated tubulin (bottom arrow), which increased during neuronal differentiation.Primary cortical neurons were differentiated from 4–22 days (TIF)Click here for additional data file.


The cyclic GMP-AMP synthase (cGAS), upon cytosolic DNA stimulation, catalyzes the formation of the second messenger 2′3′-cGAMP, which then binds to stimulator of interferon genes (STING) and activates downstream signaling. It remains to be elucidated how the cGAS enzymatic activity is modulated dynamically. Here, we reported that the ER ubiquitin ligase RNF185 interacted with cGAS during HSV-1 infection. Ectopic-expression or knockdown of RNF185 respectively enhanced or impaired the IRF3-responsive gene expression. Mechanistically, RNF185 specifically catalyzed the K27-linked poly-ubiquitination of cGAS, which promoted its enzymatic activity. Additionally, Systemic Lupus Erythematosus (SLE) patients displayed elevated expression of RNF185 mRNA. Collectively, this study uncovers RNF185 as the first E3 ubiquitin ligase of cGAS, shedding light on the regulation of cGAS activity in innate immune responses. Ubiquitination has been demonstrated to serve as an effective means to catalyze the rapid, dynamic and versatile regulatory processes that are activated when hosts face microbes. Given the critical functions of cytosolic DNA sensing pathway in anti-viral innate immune responses and the pathogenesis of autoimmune diseases, they are subjected to manifold spatial and temporal modulations shaping the strength and duration of the signaling pathways. Recent progress has characterized the cyclic GMP-AMP synthase (cGAS) as the primary DNA sensor that initiates stimulator of interferon genes (STING)-dependent signaling pathway. However, it remains poorly understood how cGAS activity is modulated dynamically. In this study, we identify E3 ubiquitin ligase RNF185 as a positive regulator of cGAS-STING signaling. Knockdown of RNF185 significantly attenuates IRF3-responsive gene expression. ER-resident RNF185 interacts with cGAS and catalyzes the K27-linked poly-ubiquitination of cGAS upon HSV-1 challenges, which thus potentiates cGAS enzymatic activity. Notably, systemic lupus erythematosus (SLE) patients have elevated expression of RNF185 mRNA. Our study uncovers RNF185 as the first E3 ubiquitin ligase of cGAS and suggests RNF185 as an important target for modulating antiviral response. The innate immune system serves as the first line of host defense against invading microbes. Upon recognition by an array of host germline-encoded pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and DNA sensors, microbial nucleic acids trigger the initiation of intracellular signaling cascades that lead to the induction of type I interferons as well as pro-inflammatory cytokines, which are a prerequisite for eliciting immediate antiviral responses and adaptive immunity to ultimately eradicate the infection ,2,3,4.in vivo [Listeria monocytogenes and Mycobacterium tuberculosis) infection [-/- mice are more vulnerable to lethal infection after exposure to herpes simplex virus 1 (HSV-1) than wild-type mice [Microbial RNA-sensing machinery and the corresponding downstream signaling cascade have been well characterized during the past decade, whereas the microbial DNA sensing represents a fast evolving field for understanding the corresponding innate immune signaling pathways ,6,7,8,9.in vivo . Analysenfection ,18,19,20ype mice .Notably, cGAS possesses nucleotidyl transferase activity, converting ATP and GTP into noncanonical cyclic dinucleotide 2′3′-cGAMP in the presence of DNA ,22. As aProtein post-translational modifications, such as phosphorylation, ubiquitination, and SUMOylation, are central to the host innate immune regulations ,28. cGASThe E3 ubiquitin ligase RNF185 potentially modulated the osteogenesis or protein quality control on the ER ,36,37. IRnf185 siRNA 1# and mouse Rnf185 siRNA 2#)  in L929 cells, stimulated by the herring testis DNA (HT-DNA) transfection  transfection  . As expesfection  or the Dsfection  or RNA vRnf185 in Raw264.7 cells also significantly attenuated the expression of the IRF3-responsive genes , when challenging cells with HSV-1  in BMDMs stimulated by HSV-1  was measured after HT-DNA stimulation. As shown in Ifnb, Ifna4 and Cxcl10 was restored by rRNF185 WT, but not rescued by rRNF185 C39A. These data suggest that RNF185 potentially modulates the cytosolic DNA sensing pathway depending on its enzymatic activity.To rule out potential off-target effects of the RNF185 siRNA, we generated two RNA interference (RNAi)-resistant RNF185 constructs, named rRNF185 WT and rRNF185 C39A, in which silent mutations were introduced into the sequence targeted by the siRNA without changing the amino acid sequence of the corresponding proteins. L929 cells were first transfected with control or RNF185 siRNA followed by introduction of control or indicated rRNF185 plasmids, respectively. Then the induction of IRF3-responsive genes (RNF185 as well as ISG15 and OASL-1 (type I IFNs inducible genes) in peripheral blood mononuclear cells (PBMCs) isolated from SLE patients and healthy controls by QPCR analysis. The RNF185 mRNA expression was significantly up-regulated in SLE patients as compared with healthy controls  induced by cGAMP  assays. As expected, HA-tagged RNF185 associated with Flag-tagged cGAS . It was A series of deletion mutants of cGAS and RNF185 were employed to map the domains responsible for RNF185-cGAS interaction . Notably, the modification reappeared when K27, rather than other lysines, was reintroduced into the ubiquitin K0 mutant . MoreoveCxcl10 and Ifnb mRNAs as well as the phosphorylation of TBK1 and IRF3 triggered by cGAS K173R/384R mutant were much lower than by wild-type cGAS in L929 cells stimulated with or without HT-DNA  to arginine (R) mutation scanning. When the two lysines (K173 and 384) on cGAS were all mutated to arginines, the poly-ubiquitination of cGAS was almost completely abolished . To subst HT-DNA .Rnf185 knockdown L929 cells  promoted recombinant cGAS to produce much more 2′3′-cGAMP than the rRNF185 C39A mutant  represents one of the immediate responses to cytosolic DNA virus infections. ELISA assays indicated that the production of IFN-β was markedly reduced in h HT-DNA . Standar HT-DNA  Site-directed mutagenesis revealed that lysines 173 and 384 on cGAS were major acceptor sites of the polyubiquitin chain. (d) RNF185 specifically catalyzed the K27-linked polyubiquitin chain on cGAS. (e) DNA virus infection induces the ubiquitination of endogenous cGAS by RNF185, as evidenced by the observation that RNAi-mediated silencing of RNF185 diminished these effects. (f) The ubiquitination of cGAS potentiates its enzymatic activity and boosts the production of cGAMP. Taken together, RNF185 is an authentic E3 ubiquitin ligase for cGAS and promotes its activation.RNF185 was previously shown to catalyze the ubiquitin-mediated degradation of several proteins  and modulate the protein quality control on ER ,36,37. IIt is recently well established that the aberrant activation of the cGAS-STING signaling by self-DNA causes severe autoimmune or auto-inflammatory disorders, such as SLE ,39,47. WcGAS-STING signaling is essential for monitoring mitochondrial DNA (mtDNA) released into cytoplasm during mitochondrial membrane permeabilization or stress ,49,50. IUbiquitination is a versatile post-translational modification critical in innate immunity ,54. DiffEthical approval for this study was granted by the Clinical Research Ethics Committee of Zhongshan Hospital, Fudan University School of Medicine. All the participants gave written informed consent before enrollment. Age matched 32 healthy volunteers were recruited as controls. All healthy volunteers used as controls also provided written informed consent.We collected 34 patients all fulfilling the American College of Rheumatology classification criteria for SLE . These pPeripheral blood (8 ml) was sampled in 10ml EDTA containing Vacutainer K2E (BD biosciences). Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation using Ficoll-Paque PLUS  and the RNA was extracted from PBMCs using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions.L929 cells (ATCC) were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS and 1% penicillin-streptomycin. HEK293T (ATCC), HEK293FT (kindly provided by Ke Lan’ lab in Shanghai Pasteur Institute), MEFs (ATCC) and RAW264.7 cells (ATCC) were maintained in DMEM plus 10% FBS (Gibco), supplemented with 1% penicillin-streptomycin (Invitrogen). Vero cells (kindly provided by Ke Lan’ lab in Shanghai Pasteur Institute) were cultured in MEM (SAFC Biosciences) supplemented with 10% FBS and 1% penicillin-streptomycin. BMDMs (bone marrow derived macrophages) were prepared as described previously . HEK293TThe individual SMART pool siRNA probes (Dharmacon) against a mouse ubiquitin-E3-ligase sub-library of 43 genes encoding RING finger proteins were transfected into L929 cells. 48h after transfection, cells were left uninfected or infected with HSV-1 for 6h, and then cells were directly collected into lysis buffer and cDNA was obtained according to the manufacturer's instruction . The quantifications of gene transcripts were performed by real-time PCR.™  following transfection with pCMV-Zeo-STING [HEK293T/STING cells were originated from HEK293T cells selected by Zeocin eo-STING . L929/cGRNF185, cGAS, STING, TBK1, IRF3, AMFR, Trim32, Trim56, DDX41, DAI, IFI16 cDNAs were obtained by standard PCR techniques from thymus cDNA library and subsequently inserted into mammalian expression vectors as indicated. pCMV-Zeo-STING was kindly provided by Fanxiu Zhu . pET21b-PFO 85–1500 a.a. was a gift from Zhengfan Jiang . The reporter plasmids (IFNβ-luciferase and pTK-Renilla) have been described previously . All poiThe rabbit polyclonal antibody against cGAS was from Cell Signaling Technology and Sigma-Aldrich. The polyclonal antibody against RNF185 was from Abcam. The antibodies against hemagglutinin (HA), Myc, GFP, and ubiquitin were purchased from Santa Cruz Biotechnology. Mouse monoclonal Flag antibody and β-actin antibody were obtained from Sigma-Aldrich. The TBK1 antibody was from Abcam. The IRF3 antibody was from Santa Cruz Biotechnology. Phospho-TBK1 and Phospho-IRF3 antibody was from Cell Signaling Technology. The antibodies against CoxIV, Calreticulin and Calnexin were from Abcam. Anti-Flag (M2)-agarose was from Sigma-Aldrich.Herring testis (HT) DNA was from Sigma. Salmon sperm DNA was from TREVIGEN. Poly(I:C) was purchased from Invivogen. IFNα2a was from PBL Assay Science. cGAMP was obtained from InvivoGen. In some experiments, cGAMP was delivered into cultured cells by digitonin permeabilization method as previously described .Chemically synthesized 21-nucleotide siRNA duplexes were obtained from Invitrogen and Gene-Pharma, and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. RNA oligonucleotides used in this study are as follows:N.C.: 5-UUC UCC GAA CGU GUC ACG UTT-3;RNF185 #1: 5′- AAU CUU CCC UGG AAG CUU UTT-3′;RNF185 #2: 5′- GCC ACA GCA UUU AAC AUA ATT -3′.Luciferase reporter assays were performed as described previously .Total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and then subjected to reverse transcription with PrimeScript RT Master Mix (Takara). The quantifications of gene transcripts were performed by real-time PCR using Power SYBR GREEN PCR MASTER MIX (ABI). GAPDH served as an internal control. PCR primers used to amplify the target genes were shown as follows:Gapdh: sense (5′-GAA GGG CTC ATG ACC ACA GT-3′), antisense (5′-GGA TGC AGG GAT GAT GTT CT-3′); Rnf185: sense (5′-AGC AGA CTG GGA TTG TCT TG-3′); antisense (5′-CCA TTG CTG CTG CCA CTG GG -3′); Ifnb: sense (5′-AGA TCA ACC TCA CCT ACA GG-3′), antisense (5′-TCA GAA ACA CTG TCT GCT GG-3′); Ifna4: sense (5′-ACC CAC AGC CCA GAG AGT GAC C-3′), antisense (5′-AGG CCCT CTT GTT CCC GAG GT-3′); Cxcl10: sense (5′-CCT GCC CAC GTG TTG AGA T-3′), antisense (5′-TGA TGG TCT TAG ATT CCG GAT TC-3′); GAPDH: sense (5′-CGG AGT CAA CGG ATT TGG TC-3′), antisense (5′-GAC AAG CTT CCC GTT CTC AG-3′); RNF185: sense (5′-AGG ACC CCA GAG AGA AGA CC -3′), antisense (5′-CAA TTC CAA AAG ACA TCT GG-3′); IFNB: sense (5′-ATT GCC TCA AGG ACA GGA TG-3′), antisense (5′-GGC CTT CAG GTA ATG CAG AA-3′); IFNA2: sense (5′-CCT GAT GAA GGA GGA CTC CAT T-3′), antisense (5′-AAA AAG GTG AGC TGG CAT ACG-3′); IFNA5: sense (5′-TCC TCT GAT GAA TGT GGA CTC T-3′), antisense (5′-GTA CTA GTC AAT GAG AAT CAT TTC G-3′); ISG15: sense (5′-GAG AGG CAG CGA ACT CAT CT-3′), antisense (5′-CTT CAG CTC TGA CAC CGA CA-3′); OASL-1: sense (5′-CCA TCA CGG TCA CCA TTG TG-3′), antisense (5′-ACC GCA GGC CTT GAT CAG-3′).Two-step immuno-precipitation and ubiquitination assays were performed as described previously . For theFor the second-round immunoprecipitation assay, the immunoprecipitates were denatured by heating for 5 min in the Lysis buffer containing 1% SDS. The elutes were diluted by 10-fold with Lysis buffer followed by reimmunoprecipitating with the anti-Flag (M2)-agarose. After extensive wash, the immunoprecipitates were subjected to immunoblot analysis.For denaturing immunoprecipitation, cells were lysed in 1% SDS buffer  and denatured by heating for thirty minutes. The lysates were centrifuged and diluted with Lysis buffer  until the concentration of SDS was decreased to 0.1%. The diluted lysates were immunoprecipitated with the indicated antibodies for four hours to overnight at 4°C before adding protein A/G agarose for two hours. After extensive wash, the immunoprecipitates were subjected to immunoblot analysis.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as previously described.Native gel electrophoresis for IRF3 dimerization was carried out as described previously .6 L929 cells in a 12.5 μl reaction containing 2 mM ATP, 1 U/μl of Benzonase and 2 ng/μl of PFO for 1.5 hr at 30°C. The cells were then subjected to the Native PAGE assay.The cGAMP activity assay was performed as described previously . Briefly2, 2 mM ATP, 2 mM DTT. The reaction was carried out at 37°C for 1 hr and then resolved by SDS-PAGE. Ubiquitinated products were detected by immunoblotting with indicated antibodies.For the synthesis of polyUb chains, purified GST-RNF185 or mutants, His-Flag-cGAS, was incubated with E1 (50 nM), E2 (0.3 mM), ubiquitin (10 μM) (Boston Biochem) in a reaction buffer containing 50 mM Tris–HCl, pH 7.5, 5 mM MgClRNF185 WT or RNF185 C39A catalyzed ubiquitinated cGAS or mutant was mixed with reaction buffer  in the presence of Salmon sperm DNA. After incubation at 37°C for 45 min, the samples were centrifuged at 16,000 × g for 10 min. The product in the supernatant was separated from cGAS and DNA by passing through a 10kD ultrafiltration filter (Millipore). The samples were diluted by 5-fold and loaded onto a MonoQ ion exchange column  equilibrated with the running buffer (50 mM Tris-HCl pH 8.5) and eluted with a NaCl gradient of 0 to 0.5 M in the running buffer.HSV-1 and HSV-1-GFP were kindly provided by Dr. Wentao Qiao  and Dr. Chunfu Zheng , respectively. HSV-1 was propagated and titered by plaque assays on Vero cells. SeV replication were measured as viral RNA expression using quantitative PCR with the following specific primers: sense (5′- GCT GCC GAC AAG GTG AGA GC -3′), antisense (5′- GCC CGC CAT GCC TCT CTC TA -3′). The MEF cells infected by HSV-1-GFP were quantified using a FACS Calibur (BD Biosciences) and the data was analyzed using Flowjo software (Tree Star).Concentrations of the cytokine in culture supernatants were measured by VeriKine Kit (PBL Assay Science) according to the manufacturer's instructions.Confocal microscopy was performed as previously described . BrieflyMitochondria and ER membranes were purified on discontinuous sucrose gradients as previously described, with some modifications ,68. BrieStudent’s t test was used for the statistical analysis of two independent treatments. The difference in RNF185 mRNA level, ISG15 mRNA level or OASL-1 mRNA level between subject groups was analyzed by Mann-Whitney test. For all tests, a P value of < 0.05 was considered statistically significant.S1 FigHomo sapiens), RNF185 (Mus musculus), RNF5 (Homo sapiens), and RNF5 (Mus musculus).(A) Amino acid sequence alignment by the DNAMAN software was shown for RNF185 ((TIF)Click here for additional data file.S2 FigIfnb, Ifna4 and Cxcl10 mRNAs using quantitative PCR. (B) PBMCs were stimulated with IFNα 2a in different dose gradients for 16h, followed by assessing the induction of RNF185, ISG15 and OASL-1 mRNAs using quantitative PCR. (C) PBMCs were stimulated with IFNα 2a (1×103 U/ml) in early time points (2h and 4h), followed by assessing the induction of RNF185, ISG15 and OASL-1 mRNAs using quantitative PCR. (D) PBMCs were stimulated with the 25% serum or 50% serum from healthy donors and SLE patients for 12h. Induction of RNF185, ISG15 and OASL-1 mRNAs was measured by quantitative PCR. (E) PBMCs were stimulated with the 25% serum or 50% serum from healthy donors and SLE patients in early time points (2h and 4h). Induction of RNF185, ISG15 and OASL-1 mRNAs was measured by quantitative PCR. Data from A-E are presented as means ± S.D. from three independent experiments. *, P < 0.05; **, P < 0.01. n.s., not significant.(A) The indicated siRNAs were transfected into L929 cells. Forty-eight hours later, cells were stimulated with HT-DNA or poly(I:C), followed by assessing the induction of (TIF)Click here for additional data file.S3 FigIfnb, Ifna4 and Cxcl10 mRNAs was measured by quantitative PCR after HSV-1 (MOI = 5) invasion (A) or SeV infection (B) for 6h. (C) BMDMs were transfected with the negative control (N.C.) or Rnf185 siRNAs. Cell lysates were immunoblotted with the indicated antibodies. (D and E) The indicated siRNAs were transfected into BMDMs. Induction of Ifnb, Ifna4 and Cxcl10 mRNAs was measured by quantitative PCR after HSV-1 (MOI = 5) invasion (D) or SeV infection (E) for 6h. (F) BMDMs transfected with the indicated siRNAs were infected with HSV-1 (MOI = 5) or SeV (50 HAU/ml) for 36h. The titer of HSV-1(left panel) was determined by standard plaque assay, and SeV (right panel) replication was determined by detection of SeV RNA by quantitative PCR. Data from A, B, D-F are presented as means ± S.D. from three independent experiments. **, P < 0.01. n.s., not significant.(A and B) The indicated siRNAs were transfected into RAW264.7 cells. Induction of (TIF)Click here for additional data file.S4 FigRnf185 siRNA. 48h after transfection, cells were treated with or without HT-DNA. Induction of Ifnb mRNA was measured by quantitative PCR. Data are presented as means ± S.D. from three independent experiments. **, P < 0.01. (D) L929/cGAS cells were transfected with the negative control (N.C.) or Rnf185 siRNA. 48h after transfection, cells were treated with or without HT-DNA, and cell extracts were analyzed for the phosphorylation of TBK1 and IRF3.(A and B) The indicated siRNAs were transfected into L929 cells. Forty-eight hours after transfection, cells were treated with HT-DNA (A) or poly(I:C) (B) for the indicated time periods, and cell extracts were analyzed for TBK1 phosphorylation. (C) L929/cGAS cells were transfected with the negative control (N.C.) or (TIF)Click here for additional data file.S5 FigIfnb and Cxcl10 mRNAs was measured by quantitative PCR. (D) L929/cGAS WT cells and L929/cGAS K173/384R cells were transfected with or without HT-DNA, and cell extracts were analyzed for the phosphorylation of TBK1 and IRF3. Data from A and C are presented as means ± S.D. from three independent experiments. **, P < 0.01.(A) Quantification of colocalization of RNF185 and cGAS in (TIF)Click here for additional data file.S6 FigIfnb mRNA was measured by quantitative PCR after HSV-1 (MOI = 0.5) infection for 6h. The proteins with at least a 2-fold decrease compared to the control were defined as the positive candidates (shown in red): RNF185, RNF45 (a.k.a. AMFR), RNF128.The indicated individual siRNA oligos were transfected into L929 cells. Induction of (TIF)Click here for additional data file.


Epithelial ovarian cancer is the leading cause of death among gynaecological cancers. Previous studies have demonstrated that epidermal growth factor receptor (EGFR) ligands can induce ovarian cancer cell invasion by down-regulating E-cadherin. Betacellulin is a unique member of the EGF family. It is overexpressed in a variety of cancers and is associated with reduced survival. However, the biological functions and clinical significance of betacellulin in ovarian cancer remain unknown. In the current study, we tested the hypothesis that betacellulin induces ovarian cancer cell migration by suppressing E-cadherin expression. Treatment of SKOV3 and OVCAR5 ovarian cancer cell lines with betacellulin down-regulated E-cadherin, but not N-cadherin. In addition, betacellulin treatment increased the expression of Snail and Slug, and these effects were completely blocked by pre-treatment with EGFR inhibitor AG1478. Interestingly, only knockdown of Slug reversed the down-regulation of E-cadherin by betacellulin. Betacellulin treatment induced the activation of both the MEK-ERK and PI3K-Akt signaling pathways, and it also significantly increased ovarian cancer cell migration. Importantly, the effects of betacellulin on E-cadherin, Slug and cell migration were attenuated by pre-treatment with either U0126 or LY294002. Our results suggest that betacellulin induces ovarian cancer migration and Slug-dependent E-cadherin down-regulation via EGFR-mediated MEK-ERK and PI3K-Akt signaling. Ovarian cancer is the fifth most common cause of cancer-related death in women and the leading cause of death from gynaecological malignancies . OvarianE-cadherin, also known as cadherin 1 (CDH1), is a classical transmembrane cell-cell adhesion glycoprotein and a well-known tumor suppressor. E-cadherin plays an important role in maintaining normal epithelial cell polarity and structure , 19. DowOur previous studies have shown that EGF induces ovarian cancer cell migration and invasion by down-regulating E-cadherin expression through a variety of signaling pathways –29. WherP=0.0502, median 15.54 vs. 18.1 months; P=0.481; To investigate the potential clinical relevance of BTC in ovarian cancer, we queried 489 ovarian cancers from The Cancer Genome Atlas (TCGA ) for up-Next, we examined the effects of BTC on E-cadherin and N-cadherin expression in two ovarian cancer cell lines (SKOV3 and OVCAR5). As shown in Figure To investigate the involvement of Snail, Slug and/or Twist in BTC-induced E-cadherin down-regulation, we first examined the time-dependent effects of BTC on their mRNA and protein levels in SKOV3 cells. Whereas BTC treatment did not alter Twist mRNA levels  mixture of M199/MCDB105 medium (Sigma-Aldrich) supplemented with 10% (vol/vol) fetal bovine serum  and 1% (vol/vol) penicillin/streptomycin (Gibco). Cells were cultured at 37°C in a humidified atmosphere containing 5% COE. coli-derived recombinant human betacellulin (Asp32-Tyr111) was obtained from R&D Systems. Human recombinant epidermal growth factor (E9644), AG1478 and LY294002 were obtained from Sigma-Aldrich. U0126 was obtained from Calbiochem.The monoclonal antibodies used in this study were: anti-human E-cadherin , anti-human N-cadherin , anti-porcine α-tubulin , anti-human Snail , and anti-human phospho-ERK1/2 (Thr202/Tyr204) , anti-human Slug . The polyclonal antibodies used were: anti-rat ERK1/2 , anti-human phospho-Akt , anti-mouse Akt . The horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories. plusSMARTpool siRNA (Dharmacon) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. ON-TARGETplus non-targeting control pool siRNA  was used as a transfection control in all experiments.To knock down endogenous Snail or Slug, cells were plated at low density, allowed to recover for 24 hours, and then transfected with 50 nM ON-TARGETCDH1), 5′-ACA GCC CCG CCT TAT GAT T-3′ (sense) and 5′-TCG GAA CCG CTT CCT TCA-3′ (antisense); N-cadherin (CDH2), 5′-GGA CAG TTC CTG AGG GAT CA-3′ (sense) and 5′-GGA TTG CCT TCC ATG TCT GT-3′ (antisense); Snail (SNAI1), 5′-CCCCAATCGGAAGCCTAACT-3′ (sense) and 5′-GCTGGAAGGTAA ACT CTG GAT TAG A-3′ (antisense); Slug (SNAI2), 5′-TTC GGACCC ACA CAT TAC CT-3′ (sense) and 5′-GCAGTGAGGGCAAGA AAA AG-3′ (antisense); Twist (TWIST1), 5′-GGA GTC CGC AGT CTT ACG AG-3′ (sense) and 5′-TCT GGA GGA CCT GGT AGA GG-3′ (antisense); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GAG TCA ACGGAT TTG GTC GT-3′ (sense) and 5′-GAC AAG CTT CCC GTTCTC AG-3′ (antisense). The amplification parameters were 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. At least three separate experiments were performed on different cultures and each sample was assayed in triplicate. A mean value was used for the determination of mRNA levels by the comparative Cq method (2−ΔΔCq) with GAPDH as the reference gene.Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription was performed with 3 μg of RNA, random primers, and Moloney murine leukemia virus reverse transcriptase (Promega). SYBR Green RT-qPCR was performed on Applied Biosystems 7300 Real-Time PCR System equipped with 96-well optical reaction plates. Each 20 μl RT-qPCR reaction contained 1×SYBR Green PCR Master Mix (Applied Biosystems), 20 ng cDNA and 150 nM of each specific primer. The primers used were: E-cadherin (g for 10 minutes at 4°C and supernatant protein concentrations were quantified using the DC Protein Assay (Bio-RadLaboratories) with BSA  as the standard. Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking with Tris-buffered saline containing 5% non-fat dry milk for 1 hour, membranes were incubated overnight at 4°C with primary antibodies E-cadherin (1:3000), N-cadherin (1:3000), α-Tubulin (1:3000), phospho-ERK1/2 (1:3000), ERK1/2 (1:3000), phospho-Akt (1:3000), Akt (1:3000), Snail (1:1000) or Slug (1:1000), followed by incubation with the peroxidase-conjugated secondary antibody (1:5000). Immunoreactive bands were detected with enhanced chemiluminescent or SuperSignal West Femto substrate (Pierce). Membranes were stripped with stripping buffer  at 50°C for 30 minutes and reprobed with anti-α-Tubulin, anti-ERK1/2 or anti-Akt as a loading control. Immunoreactive band intensities were quantified by densitometry, normalized to those of the relevant loading control, and the results are expressed as fold change relative to the respective control.Cells were lysed in lysis buffer  containing protease inhibitor cocktail (Sigma-Aldrich). Lysates were centrifuged at 20,000×5 cells in 250 μL of medium with 0.1% FBS. Medium with 10% FBS (750 μl) was added to the lower chamber and served as a chemotactic agent. After 12 hours incubation, non-migrating cells were wiped from the upper side of the membrane and cells on the lower side were fixed in cold methanol (−20°C) and air dried. Cells were stained with Crystal Violet and counted using a light microscope (10× objective) equipped with a digital camera (QImaging) and Northern Eclipse 6.0 software. Five microscopic fields were counted per insert, triplicate inserts were used for each individual experiment, and each experiment was repeated at least three times.Migration assays were performed in Boyden chambers with minor modifications . TransweP < 0.05 and are indicated by different letters.Results are presented as the mean ± SEM of at least three independent experiments. PRISM software (GraphPad Software Inc.) was used to perform one-way ANOVA followed by Tukey's multiple comparison test. Means were considered significantly different if


Bax, and Bax/Bcl2 ratio in cultured podocytes. These results indicate that natriuretic peptide exerts a renoprotective effect via inhibiting phosphorylation of p38 MAPK in podocytes.Guanylyl cyclase-A (GC-A) signaling, a natriuretic peptide receptor, exerts renoprotective effects by stimulating natriuresis and reducing blood pressure. Previously we demonstrated massive albuminuria with hypertension in uninephrectomized, aldosterone-infused, and high salt-fed (ALDO) systemic GC-A KO mice with enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) in podocytes. In the present study, we examined the interaction between p38 MAPK and GC-A signaling. The administration of FR167653, p38 MAPK inhibitor, reduced systolic blood pressure (SBP), urinary albumin excretion, segmental sclerosis, podocyte injury, and apoptosis. To further investigate the local action of natriuretic peptide and p38 MAPK in podocytes, we generated podocyte-specific (pod) GC-A conditional KO (cKO) mice. ALDO pod GC-A cKO mice demonstrated increased urinary albumin excretion with marked mesangial expansion, podocyte injury and apoptosis, but without blood pressure elevation. FR167653 also suppressed urinary albumin excretion without reducing SBP. Finally, we revealed that atrial natriuretic peptide increased phosphorylation of MAPK phosphatase-1 (MKP-1) concomitant with inhibited phosphorylation of p38 MAPK in response to MAPK kinase 3 activation, thereby resulting in decreased mRNA expression of the apoptosis-related gene,  Aldosterone has attracted much attention in the process of renal injury leading to proteinuria and chronic renal failureNatriuretic peptides consist of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP and BNP exert physiological effects such as diuresis and vasodilation through guanylyl cyclase-A (GC-A)/natriuretic peptide receptor-A (NPR-A)1012In the present study, we generated podocyte-specific (pod) GC-A conditional KO (cKO) mice and then examined the effects of p38 MAPK in systemic and pod GC-A cKO mice administered aldosterone, uninephrectomy, and a high salt diet.All ALDO mice were uninephrectomized and administered aldosterone and a 6% high salt diet for four weeks. First, we examined p38 MAPK phosphorylation in ALDO wild-type and systemic GC-A KO mice . PhosphoWe examined renal histology at four weeks after aldosterone infusion. A mild mesangial expansion in superficial glomeruli was observed in ALDO wild-type mice . In contTgfb1) is a profibrotic growth factor and is closely associated with matrix accumulation and renal fibrosis. Analysis of glomerular expression revealed increased Tgfb1 mRNA levels in ALDO systemic GC-A KO mice, and this upregulation was significantly reduced by FR167653 (Ccl2 (MCP1) is one of macrophage chemoattractant molecules, and Adgre1 (F4/80) is one of markers of macrophages. Expression of Ccl2 (MCP-1) mRNA was increased in ALDO systemic GC-A KO mice, and administration of FR167653 significantly reduced Ccl2 mRNA levels (Adgre1 (F4/80) mRNA expression in ALDO systemic GC-A KO mice (Cybb (NOX-2) mRNA expression was observed  GC-A conditional KO (cKO) mice by crossing Nephrin-Cre mice with GC-A floxed mice, and then administering FR167653. Vehicle (VEH)-treated mice were also uninephrectomized, and administered 6% high salt diet and ethanol alone instead of ethanol-containing aldosterone. GC-A floxed mice without Cre recombinase were used as control mice. VEH control and pod GC-A cKO mice had normal SBP . ALDO coAt basal levels, urinary albumin excretion was not different between VEH control mice and pod GC-A cKO mice  in VEH pod GC-A cKO mice was decreased by 40% compared with that in VEH control mice  is one of main extracellular matrix in the kidney and these gene expression is relevant to extracellular matrix accumulation. The expression of extracellular matrix-related gene, such as Tgfb1 and Fn1, was enhanced with administration of aldosterone both in control mice and pod GC-A cKO mice, and was ameliorated by treatment with FR167653 (Ccl2 (MCP1) is one of macrophage chemoattractant molecules, and Adgre1 (F4/80) is one of markers of macrophages. Expression patterns of Ccl2 and Adgre1 were similar to expression levels of Tgfb1 and Fn1 (Cybb and Trp53 (p53) mRNA levels in both control mice and pod GC-A cKO mice, and FR167653 ameliorated these changes  or constitutive active MKK3 (CA) . Phosphodose ANP . Consistodocytes . As a rewith ANP . To confwith ANP . Adminiswith ANP , indicatin vitroThe present study demonstrated that blockade of p38 MAPK ameliorates glomerular injury in ALDO systemic GC-A KO mice. As we previously reported, ALDO wild-type mice show mild albuminuria, a slight increase in mesangial area, and mild podocyte injury; however, ALDO systemic GC-A KO mice show massive albuminuria with prominent mesangial expansion and podocyte injuryet al. reported that administration of FR167653 to Dahl salt-sensitive rats reduces expression of interleukin-1β and tumor necrosis factor-α in the whole kidney without altering blood pressureet al. recently reported that chronic p38 MAPK inhibition improves vascular function and reduces systolic blood pressure in angiotensin II-stimulated hypertension in miceInterestingly, administration of FR167653 reduced systolic blood pressure in ALDO systemic GC-A KO mice by 20 mmHg, although it had no effect on systolic blood pressure in ALDO wild-type mice. ALDO systemic GC-A KO mice had similar levels of serum aldosterone, and daily urinary sodium excretion, suggesting that the reduction in systolic blood pressure was not due to increased sodium excretion. Other possible explanations are changes in blood vessel tone and cardiac output. Tojo et al. reported that BNP activates phosphorylation of p38 MAPK in microvascular endothelial cellset al. reported that uninephrectomized pod GC-A cKO mice with deoxycorticosterone acetate and high salt diet exhibit similar blood pressure and increased albuminuria compared with control mice, confirming the protective role of natriuretic peptides/GC-A pathway in podocytes26Kuhn Aldosterone has been shown to phosphorylate p38 MAPK in many cell types, including vascular smooth muscle cellsdb/db mice48Bax expression, leading to decreased levels of apoptosis.Previous studies have reported that aldosterone induces apoptosis in mesangial cellsIn conclusion, inhibition of p38 MAPK pathway ameliorates glomerular injury in uninephrectomized, high-salt fed, and aldosterone-infused mice by protecting against podocyte injury. These results indicate that p38 MAPK inhibitors may represent promising therapeutic targets against renal injury.Aldosterone was obtained from Sigma Aldrich . FR167653, p38α MAP Kinase inhibitor, was kindly provided by Astellas Pharma Inc. . Primary antibodies used for immunohistochemical studies and Western blotting were goat anti-nephrin , rabbit anti-podocin (Sigma Aldrich), rabbit anti-phospho-p38 MAPK , rabbit total p38 MAPK , rabbit anti-WT1 , and rabbit anti-p53 , rabbit anti-phospho-MKP-1 , rabbit anti-phospho-MKK3 , and mouse anti-glyceraldehyde-3-phosphate dehydrogenase  antibodies. pRc/RSV Flag MKK3All animal experiments were performed in accordance with Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions and were approved by the Animal Experimentation Committee of Kyoto University Graduate School of Medicine. Male systemic GC-A KO miceNpr1fl/fl, Nephrin Cre/+mice) and control littermates . Male pod GC-A cKO mice or control mice at 12 weeks of age were uninephrectomized, and administered with 6% NaCl diet and aldosterone or vehicle using the same protocol for systemic GC-A KO mice.Construction of GC-A floxed mice and nephrin-promotor driven Cre recombinase transgenic mice were as described previously53Histological and electron microscopic examinations were performed as described previouslyin situ cell death detection kits . Paraffin-embedded renal sections were reacted with fluorescent dyed mixed solutions.Immunofluorescent studies for nephrin and podocin were performed as descried previouslyConditionally immortalized human podocytes were provided by Dr. Moin Saleem, and cultured and differentiated as described previously56Npr1, Tgfb1, Ccl2, Fn1, Cybb, Adgre1, Trp53, Bax, and Bcl2 mRNA expression levels were evaluated. Some primer and probe sets were described previouslyAdgre1 forward primer, 5′-tggtggtcataatctctgcttctg-3′; Adgre1 reverse primer, 5′-agacaggccccaggaaactc-3′; Adgre1 probe, 5′-FAM-cccgtctctgtattcaaccagcagcgatt-TAMRA-3′; Trp53 forward primer, 5′-gcttctccgaagactggatgac-3′; Trp53 reverse primer, 5′-gcagtgaggtgatggcagg-3′; Trp53 probe primer, 5′-FAM-tcacagtcggatatcagcctcgagctcc-TAMRA-3′; Bax forward primer, 5′-aactggtgctcaaggccct-3′; Bax reverse primer, 5′-cccggaggaagtccaatgtc-3′; Bax probe primer, 5′-tgcaccaaggtgccggaactgatca-TAMRA-3′; Bcl2 forward primer, 5′-gatgggatcgttgccttatgca-3′; Bcl2 reverse primer, 5′-cagtctacttcctctgtgatgttgta-3′; 5′-cagcatgatcctctgtcaagtttcctt-TAMRA-3′, Bcl2 probe primer, 5′-FAM-cagcatgatcctctgtcaagtttcctt -TAMRA-3′.Glomeruli were isolated by the graded sieving methods as described previouslyWestern blotting was performed as previously describedData are expressed as the mean ± SE. Statistical analysis was performed using ANOVA or Student’s t test as appropriate.How to cite this article: Kato, Y. et al. Natriuretic peptide receptor guanylyl cyclase-A pathway counteracts glomerular injury evoked by aldosterone through p38 mitogen-activated protein kinase inhibition. Sci. Rep.7, 46624; doi: 10.1038/srep46624 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.