Our recent studies have revealed 14-3-3ε (a direct inhibitor of Apaf-1) as a cytosolic cytochrome c target. Here we explore the cytochrome c / 14-3-3ε interaction and show the ability of cytochrome c to block 14-3-3ε-mediated Apaf-1 inhibition, thereby unveiling a novel function for cytochrome c as an indirect activator of caspase-9/3. We have used calorimetry, NMR spectroscopy, site mutagenesis and computational calculations to provide an insight into the structural features of the cytochrome c / 14-3-3ε complex. Overall, these findings suggest an additional cytochrome c-mediated mechanism to modulate apoptosome formation, shedding light onto the rigorous apoptotic regulation network.Apoptosis is a highly regulated form of programmed cell death, essential to the development and homeostasis of multicellular organisms. Cytochrome  There are two main apoptotic pathways in mammalian cells: the extrinsic or death receptor-initiated pathway and the intrinsic or mitochondria-dependent pathway3. The last is triggered by signals, including DNA damage, oxidative stress, and growth factor deprivation, which converge on the release of pro-apoptotic proteins from the mitochondria into the cytosol; one such protein is cytochrome c (Cc). Under homeostasis, Cc acts as an electron carrier in the mitochondrial respiratory chain5. Upon its release from mitochondria, Cc interacts with WD40 domains of the apoptotic protease activating factor-1 (Apaf-1) in the cytosol, enabling the assembly of the apoptosome6. This protein platform activates caspase-9 and later caspase-3, initiating the caspase cascade, which executes cell death in an orchestrated way8.Apoptosis, a specific form of programmed cell death (PCD), is an essential process for development and homeostasis of multicellular organismsc fulfills multiple roles in apoptosis, beyond Apaf-1 activation and apoptosome assembly. Indeed, Cc interacts with various cytosolic and nuclear partners along the onset of PCD10. Hence, the full scope of Cc role in apoptosis remains un-elucidated. Recently, our group has reported that Cc inhibits the histone chaperone activity of SET/TAF-Iβ in the nucleus, impairing the formation of core histone-SET/TAF-Iβ complexes under DNA damage11. However, the novel functions of cytosolic Cc stay unveiled, despite a complex network of interactions mediated by Cc during apoptosis has been suggested12.Recent findings suggest Cc and protein 14-3-3ε, a novel cytosolic Cc target under DNA damage10. This protein belongs to the 14-3-3 family14, which comprises seven conserved isoforms , arranged as homo- and heterodimers. Each monomer contains nine α-helices that form a conserved concave groove, used by 14-3-3 proteins to bind their phosphorylated targets15  when the mitogen-activated protein kinases (MAPK) cascade is active. Hence, the interaction of Cc with 14-3-3ε could modulate such inhibition somehow.Among 14-3-3ε functions, its ability to inhibit Apaf-1 stands out because it prevents apoptosome assembly and caspase activationc hinders 14-3-3ε-mediated Apaf-1 inhibition. Indeed, our results indicate a competition between Cc and 14-3-3ε for binding to Apaf-1, which enhances caspase activation. Furthermore, this new regulatory mechanism is modulated by phosphorylation of Apaf-1, which enhances its inhibition by 14-3-3ε. We further show that Cc binds to both the 14-3-3ε concave groove and the convex face, thereby providing a molecular basis for this novel modulation of apoptosome assembly.Herein, we show that Cc, we explored the interaction of Cc with 14-3-3ε when apoptosis is triggered. To this aim, HeLa cells were treated with the topoisomerase I inhibitor camptothecin (CPT), to induce DNA damage. Then, subcellular fractionation was performed, and Cc was detected in the cytosol  of cytosolic proteins associated with Cc yielded intrinsic 14-3-3ε in CPT-treated cells , which triggers the MAPK cascade, on the caspase-3 activity. Apaf-1 phosphorylation under PMA treatment was confirmed by Phos-tagTM SDS PAGE  to inhibit caspase-3 activity in non-treated and PMA-treated cell extracts. Addition of 14-3-3ε FL made extracts to exhibit significantly lower Cues Fig. . These wues Fig. . One of ues Fig. , with thues Fig. .c. Caspase-9 can indeed be phosphorylated by the ERK MAPK pathway, with the resulting modulation of its function and consequently, of caspase-3 activation26.In summary, these findings demonstrate how 14-3-3ε FL inhibits caspase activity, and how this inhibitory mechanism can be modulated by Apaf-1 phosphorylation. Nevertheless, the inhibitory effect of 14-3-3ε FL on caspase-3 could be caused by either a direct interaction with Apaf-1 or by hindering the complex between the latter and Cc and Apaf-1, as well as the ERK-mediated inhibition of caspase-9, we performed caspase-9 activity assays using a recombinant, truncated form of Apaf-1 lacking the Cc-binding WD40 domains (Apaf-1ΔWD40)27. Apaf-1ΔWD40 activates caspase-9 in a constitutive manner in the absence of Cc  experiments corroborated such a competition. Specifically, the ε-methyl group signal of Met80 in reduced Cc substantially broadened upon binding of the hemeprotein to 14-3-3ε  of 2.3 μM  experiments in Supplementary Figure c and 14-3-3ε FL. We followed Cc signals upon titration of 14-3-3ε FL, which caused an overall broadening of Cc resonances because Cc / 14-3-3ε FL complex formation  upon binding with 14-3-3ε FL  in the presence of 14-3-3ε FL . Interestingly, the signal corresponding to Val83  are involved in the interaction with Cc, 2D  HSQC were recorded along a titration of 14-3-3ε234-255 peptide onto 15N-labeled reduced Cc  of 14-3-3ε234-255 before and after the conjugation reaction was unambiguously assessed by 1D 1H NMR spectra .As a negative control, bovine serum albumin (BSA) protein was titrated into fluorinated 14-3-3εc and 14-3-3ε FL, we performed restrain-driven docking computations using HADDOCK software34. For these computations, we used the structural model of 14-3-3ε FL in its open conformation inferred from MD as the input structure . The 14-3-3ε FL residues that resulted in the impairment of one of the Cc-binding sites by ITC were used as 14-3-3ε AIRs. The 500 solutions obtained in the computation showed two main Cc-binding sites on 14-3-3ε FL dimer, in agreement with the stoichiometry inferred from ITC data. These solutions located Cc in both the concave and convex faces of 14-3-3ε; these interacting regions will be referred hereafter to as the concave and convex binding sites, respectively. The convex site resulted more populated, although some of the lowest energy solutions locate within the concave site . Nevertheless, convex clusters presented lower AIRs violations than the concave cluster.To further understand the molecular features of the interaction between Cite Fig. . Furtherite Fig. . Howeverc in the concave binding site of 14-3-3ε , as an active residue. This computation showed an increase in the number of solutions locating Cc / 14-3-3ε FL complex were obtained for computations excluding and including  cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM 6 HEK293T cells were cultured in 140 mm Petri dishes in DMEM containing FBS until 70% confluence was reached, and cells were then kept in DMEM without FBS for 4 h. In some experiments (indicated in the main text) cells were treated with 50 ng/mL phorbol-12-myristate-13-acetate  or treated with the same volume of DMSO for 120 min.For caspase-3 assays, 4.5 × 10BgIII–HindIII restriction sites. An empty pSUPER vector was used as a control. Cells were transfected with the corresponding pSUPER plasmid by calcium phosphate method, as described elsewhere42. Transfected cells were selected by adding puromycin (1.5 μg/mL) to the culture medium. Four days after transfection, cells were collected and cytosolic extracts were obtained.In order to knockdown 14-3-3ε expression in HEK293T cells, a construct containing the shRNA sequence (5′-GATCCCCTGTTCAATACTGCTGATTATTCTCCTTCAAGAGAGGAGAATAATCAGCAGTATTGAACATTTTTA-3′) was cloned into a pSUPER vector using 6 HeLa cells were seeded in 140 mm Petri dishes and treated with an apoptosis inducing agent  for 4 h. HeLa cells were fractionated into cytosolic, membrane/organelles and nuclear fractions using a ProteoExtract Subcellular Proteome Extraction Kit  following the manufacturer’s indications. The purity of subcellular fractions obtained was verified by western blot analysis, using anti-α-tubulin and anti-COX IV for detecting cytosol and mitochondria, respectively.For immunoprecipitation assays, 6 × 1043. Briefly, cells were trypsinized and collected by centrifugation (5 min at 1500×g) and washed twice with phosphate buffer saline (PBS) and once with Cellular Extract Buffer . Two volumes of CEB were added to cells, and they were incubated for 15 min on ice, allowing the hypotonic swell of cells. The cells were then disrupted by 20–25 strokes with a Douncer homogenizer and lysates were centrifuged at 15,000×g for 15 min at 4 °C to remove organelles.For caspase-3 activity assays, cytosolic extracts were obtained from HEK293T cells as previously described with briefly modificationsc. Quantity of 300 μg of cytosolic samples were incubated with 50 μL Sepharose 6B (Sigma-Aldrich) for 2 h at 4 °C under agitation as a pre-clearing step to reduce non-specific binding. Samples were centrifuged and 20 μL of rabbit anti-human cytochrome c (Cc) were added to supernatants and incubated overnight at 4 °C under rotation. As a negative control, 300 μg of cytosolic lysates were incubated with 1 μg of anti-mouse IgG under the same conditions. Afterwards, 50 μL Protein A Sepharose  was added to each lysate for 4 h at 4 °C under rotation. The protein-Sepharose complexes were washed extensively, collected by centrifugation and boiled in freshly prepared reducing loading buffer. Controls including 30 μg of cytosolic lysates were run concurrently with the immunoprecipitated (IP) samples in the western blot assays.Cytosolic fractions from subcellular fractionation of control and CPT-treated HeLa cells were used to immunoprecipitate CTM SDS PAGE (Wako) — which reduce the electrophoretic mobility of phosphorylated proteins.Apaf-1 immunoprecipitation was performed from total non-treated and PMA-treated lysates. Apaf-1 IP samples were analyzed by western blot using a Phos-tagc serum was obtained after immunizing male rabbits with full length recombinant Cc suspended in a 0.85% solution of NaCl (20 mg/mL). The suspension was then incorporated into an equal amount of complete (Freund) adjuvant obtained from Difco . Mouse monoclonal anti-α-Tubulin , as well as secondary horseradish peroxidase (HRP)-conjugated anti-mouse IgG , rabbit polyclonal anti-14-3-3ε , and anti-rabbit IgG  were obtained from Sigma-Aldrich. Rabbit polyclonal antibody to cytochrome c oxidase subunit IV (COX IV) was from Abcam . Mouse monoclonal anti-Apaf-1  was obtained from Santa Cruz Biotechnology, INC. Mouse monoclonal anti-phosphoserine (Millipore clone A4) was obtained from Merck.Rabbit anti-human Cc, α-Tubulin, COX IV, and 14-3-3ε in the subcellular fractions or IP samples, protein content was measured using the DCTM protein assay . Proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in 12% gels and then transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). Membranes were blocked in 5% non-fat dry milk in PBS with Tween-20 (TPBS) and immunoblotting was performed with primary antibodies. HRP-conjugated secondary antibodies were used for detection. The immunoreactive bands were detected using Amersham ECL Plus Western Blotting Detection Reagents .For the immunoblot detection of Cc was expressed in Escherichia coli BL21 (DE3) strain as previously described32. First, 25 mL Luria-Bertani (LB) medium, supplemented with 100 μg/mL ampicillin, were grown overnight at 37 °C under agitation. Volume of 2.5 mL of pre-culture was used to inoculate 2.5 L of LB medium. The culture was incubated at 30 °C under agitation for 24 h. Then, cells were centrifuged at 6000 r.p.m. for 10 min in an Avanti J-25 centrifuge (Beckman Coulter). Following this, cells were re-suspended in 1.5 mM borate buffer (pH 8.5), sonicated for 4 min and, finally, cellular debris was eliminated by centrifugation . For NMR measurements, 15N-labeled Cc was produced in minimal media with 15NH4Cl as a unique nitrogen source. Cc purification was carried out by ionic chromatography with a carboxy-methylcellulose matrix. Fractions containing Cc were concentrated in an Amicon (3 kDa cutoff) until correct concentration was achieved.CNdeI – XhoI restriction sites and containing a N-terminal hexahistidine tag. For cloning, the following primers were used: 5′-AGCCATATGATGGATGATCGAGAGGAT-3′ and 5′-GTGCTCGAGTCATTCCTGATTTTCGTC-3′. The 14-3-3ε core was cloned with a Stop codon introduced after residue at position 233. The primers used for this mutagenesis were 5′-ACACTATGGACTTCATAGATGCAGGGTGACGGT-3′ and 5′-ACCGTCACCCTGCATCTATGAAGTCCATAGTGT-3′. 14-3-3ε FL D21K, K50E, S59E, E92K, D99K, and S187D mutants were obtained from Mutagenex Inc. (USA). The expression of 14-3-3ε species were performed in the E. coli BL21 (DE3) strain. Cultures were grown in LB medium at 37 °C until an O.D.600 nm of 0.6–0.8 was reached. Protein expression was induced by adding 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and cultures were grown for 24 h at 30 °C. The cells were collected by centrifugation and re-suspended in 20 mM Tris-HCl (pH 8), 800 mM NaCl, 10 mM imidazole. Cells were then broken by sonication and insoluble debris was removed by centrifugation, as indicated above. 14-3-3ε species were purified by affinity chromatography, using a Ni Sepharose 6 Fast Flow  column and an imidazole gradient ranging from 10 mM to 300 mM imidazole. Protein containing fractions were concentrated in an Amicon (3 kDa cutoff) until the desired proper protein concentration was reached.14-3-3ε full length (14-3-3ε FL) was cloned into the pET-28a vector under the T7 promotor using Proteins were dialyzed against 10 mM sodium phosphate (pH 7.4) buffer for ITC measurements or 5 mM sodium phosphate (pH 6.3) buffer for NMR titrations. For caspase assays, protein was dialyzed against 20 mM HEPES (pH 7.5) buffer.234-255) was purchased from Genosphere Biotechnologies (France).14-3-3ε C-terminal peptide  at 20 °C after induction with 1 mM IPTG. Cells were broken by freeze/heat cycles and cellular debris was removed by centrifugation. The soluble fraction was purified by affinity chromatography  followed by anionic-exchange . Recombinant caspase-9 was expressed in E. coli BL21 (DE3) at 30 °C and purified as indicated above for Apaf-1ΔWD40. Protein quantification was assessed using the Bradford protein assay44.Apaf-16. Recombinant Apaf-1ΔWD40 species and 14-3-3ε FL were mixed in buffer  to a final concentration of 100 nM and 1 μM, respectively. Cc was added at different concentrations (ranging from 1 to 5 μM). After incubation for 15 min at room temperature, recombinant caspase-9 was added to a final volume of 100 μL and final concentration of 500 nM. After incubation for another 15 min at 37 °C, the caspase-9 substrate LEHD peptide coupled to 7-amino-4-trifluoromethylcoumarin  was added at a final concentration of 50 μM. The increase in fluorescence resulting from Ac-LEHD-AFC cleavage was determined in a Cary Eclipse (Varian) fluorescence spectrophotometer, using an excitation wavelength of 390 nm and an emission wavelength of 510 nm. Each experimental data were the mean ± SD value of at least three independent measurements.Caspase-9 activity was measured as previously described45. Recombinant Cc and 14-3-3ε FL were mixed and incubated with 100 μg of cytosolic HEK293T cells extracts for 60 min at 37 °C in 10 mM HEPES (pH 7.5) buffer, supplemented with 25 mM KCl, 200 μM DTT, 200 μM dATP. After incubation, 10 mM HEPES (pH 7.5), 50 mM NaCl, 40 mM β-glycerophosphate, 2 mM MgCl2, 5 mM EGTA, 0.1 mg mL−1 bovine serum albumin, and 0.1% (w/v) 3-[3-cholamidopropyldimethylammonio]-1-propanesulfonate (CHAPS) buffer was added to a final volume of 200 μL, supplemented with 40 μM of acetyl-DEVD-7-amino-4-methylcoumarin , a fluorescent substrate specific to caspase-3/7. The increase in fluorescence resulting from Ac-DEVD-AMC cleavage was determined in a Cary Eclipse (Varian) fluorescence spectrophotometer, using an excitation wavelength of 360 nm and an emission wavelength of 460 nm. To compare the effect of 14-3-3ε in different types of extracts, values for caspase-3 activity before adding recombinant 14-3-3ε FL were normalized. Each experimental data were the mean ± SD value of at least three independent measurements.In vitro caspase-3 assays were performed as formerly described with slight changesc. The reference cell was filled with distilled water. The experiments consisted of 2-μL injections of 300 μM Cc solution in 10 mM sodium phosphate buffer (pH 7.4) into the sample cell, initially containing 20 μM 14-3-3ε solution in the same buffer. All solutions were degassed before titrations were performed. Titrant was injected at appropriate time intervals to ensure the thermal power signal returned to the base line prior to the next injection. To achieve homogeneous mixing in the cell, the stirring speed was maintained constant at 750 r.p.m. The data, specifically the heat per injection normalized per mole of injectant vs. the molar ratio, were analyzed with Origin 7.0 (OriginLab Corp.). Calibration and performance tests of the calorimeter were carried out conducting CaCl2-EDTA titrations with solutions provided by the manufacturer. Control experiments (Cc solution injected into buffer) were performed in order to assess potential unspecific heat effects.All ITC experiments were performed using an Auto-ITC200  at 25 °C by titrating 14-3-3ε species and 14-3-3ε peptides with Cc with 14-3-3ε FL was followed by 1D 1H and 2D  HSQC spectra on a Bruker Avance 700 MHz at 25 °C. 50 μM of reduced, 15N-labelled Cc in 5 mM sodium phosphate buffer (pH 6.3) was titrated with unlabeled 14-3-3ε in the same buffer. 0.1 M sodium ascorbate and 10% D2O were added in order to ensure the redox state remained constant and to adjust the lock signal, respectively. Water signal was suppressed according to the WATERGATE solvent suppression method46. The interaction of reduced Cc with 14-3-3ε234-255 was performed on a Bruker Avance 600 MHz in the same way as previously described for the titration. Data were processed using TopSpin NMR 2.0 software (Bruker). Line broadening and chemical-shift perturbation analysis were performed using Sparky 3 NMR Assignment Program . CSP titration curves were fitted using the one-site binding model.NMR titration of reduced C234-255, the bioconjugation reaction was performed with 5-fluoroindole compound (Sigma) as previously described47. 500 μM acetylated 14-3-3ε234-255 peptide was mixed with 2 mM formaldehyde solution (37% w/w) and with 6 mM 5-fluoroindole for 24 h at 30 °C. Then, the sample was dialyzed against 10 mM sodium phosphate (pH 6.3) buffer overnight. Proper folding of 14-3-3ε234-255 before and after the reaction was checked by 1D 1H spectra, and the interaction with Cc was followed recording 1D 19F experiments at 25 °C on a Bruker Avance 600 MHz.To fluorinate the Lys244 side chain of 14-3-3εSedimentation velocity experiments of 14-3-3ε FL WT, D21K, K50E, S59E, E92K, D99K, and S187D were performed at 20 °C in an Optima XL-A Analytical Ultracentrifuge  with an AN50-Ti rotor. These experiments were carried out at 45,000 r.p.m. with 400-μL samples at 37 μM in 5 mM sodium phosphate buffer (pH 6.3) loaded into double sector cells. Radial scans at 280 nm were taken every 10 min and the sedimentation coefficient distribution was calculated by least squares boundary modeling of the sedimentation velocity data using the programme SEDFIT. The experimental coefficients were converted to standard conditions .234-255 peptide simulation, an unfolded and outspread conformation was used as initial coordinates. After 100 ns of simulation, the closest structure to the average from the last 40 ns of this computation was attached to the crystallographic structure of 14-3-3ε (PDB: 2BR9)15 at the C-terminal of both monomers. The resulting structure was used as the initial coordinate for the full length protein in the MD computation. With the aim to promote the opening of 14-3-3ε FL and to produce an open conformation, both consensus peptide ligands for 14-3-3 proteins included in the crystallographic structure were removed from the initial coordinates.Molecular dynamics (MD) computations were carried out using the AMBER 12 package and the AMBER-2003 force field. For the 14-3-3ε48 was used to constrain bonds involving hydrogen atoms. The PTRAJ module of AMBER was used for trajectory analysis.All simulations were carried out under periodic boundary conditions using an orthorhombic cell geometry  and PME electrostatics were set with the Ewald summation cutoff at 9 Å. Sodium counter-ions were added to neutralize the charges of the system. The structures were solvated with SPC water molecules. Afterwards, solvent and counter-ions were subjected to 2000 steps of energy minimization followed by 300 ps NPT-MD computations using isotropic molecule position scaling and a pressure relaxation time of 1 ps at 298 K. The density of the system reached a plateau during the first 150 ps. Then, the whole system was energy minimized and submitted to NVT MD computations at 298 K. The SHAKE algorithm51, using the 3ZCF.pdb structure of Cc and the resulting open model of 14-3-3ε FL from MD calculations. Dielectric constant was set as distance-dependent. Scaling of ambiguous interaction restraints (AIRs) were fixed to 0.1 for rigid body and to 1.0 for both semi-flexible simulated annealing and final refinement. AIRs for the docking simulation were generated using standard criteria. Random exclusion of AIRs was not employed. Cc residues labeled as active were those showing 1H ΔΔν1/2Binding ≥ 59.2 Hz and a solvent accessibility larger than 50% calculated with NACCESS. These active residues were Gln16, Gln42, Ser47, Ala50, Gly77, Lys79, Ile81, and Val83. Cc residues located at <4 Å from the active ones and showing high-solvent accessible surface (>50%) were labeled as passive residues. 14-3-3ε residues labeled as active were those whose mutation resulted in the impairment of one of the Cc-binding sites: Asp21, Lys50, Ser59, and Glu92. 14-3-3ε residues located at <4 Å from the active ones and showing high-solvent accessible surface (>50%) were labeled as passive residues. For each run, 10,000 rigid-body solutions were generated by energy minimization. The 1000 structures with the lowest AIRs energies were subjected to semi-flexible simulated annealing. The first 500 structures were submitted to a final refinement in explicit water. The 500 best structures were analyzed using standard criteria. AIR violations were calculated using as reference the solution with best HADDOCK score. Structures were represented using UCSF Chimera52.Restrained docking calculations were performed with the High Ambiguity Driven Docking approach (HADDOCK)Dynamic light scattering experiments were performed to evaluate the dimerization state of 14-3-3ε FL WT and D21K, K50E, S59E, E92K, D99K, and S187D mutants, in 5 mM sodium phosphate at pH 6.3. DLS experiments on 0.1 mg/mL of each 14-3-3ε species were conducted at 25 °C in a Zetasizer Nano ZS .t-test. A p-value of <0.0002 was considered significant.Statistical analysis was performed using GraphPad Prism 7 Software and experimental data of at least three independent measurements. Statistical significance was estimated using Student's Supplementary Information


Arsenite is a known carcinogen and its exposure has been implicated in a variety of noncarcinogenic health concerns. Increased oxidative stress is thought to be the primary cause of arsenite toxicity and the toxic effect is thought to be linear with detrimental effects reported at all concentrations of arsenite. But the paradigm of linear dose response in arsenite toxicity is shifting. In the present study we demonstrate that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. Taken together, these results suggest a nonlinear dose-response characteristic of arsenite with low-dose arsenite promoting adaptive responses in a process known as mitohormesis, with transient increase in ROS levels acting as transducers of arsenite-induced mitohormesis. Inorganic arsenite is a naturally occurring metalloid that is ubiquitously present in the environment. In the world millions of people are exposed to arsenite through ingestion of contaminated drinking water . ChronicThe liver has long been identified as a target organ of arsenic exposure –20. Its The purpose of the current study was an attempt to define the broad components of arsenite nonlinear effects in human and mouse primary hepatocytes and explore the role of mitochondria in this process. Primary hepatocytes were chosen over in vivo studies because of the transient, variable, and uncertain nature of tissue exposure in in vivo studies that might be of little value for evaluating dose response for mechanistic studies.2.The human hepatoma cell line HepG2 was purchased from American Type Culture Collection . Cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin-streptomycin, and 2 mM L-glutamine at 37°C in a humidified atmosphere of 95% air and 5% COPrimary mouse hepatocytes were isolated and cultured as described previously .g for 5 min at 4°C. The isolated hepatocytes were plated on collagen coated 24- and 96-well plates (Becton-Dickinson). The plating densities for mouse hepatocytes were 0.5 × 106 and 0.4 × 106 viable cells/well for 24- and 96-well plates, respectively. Human hepatocytes that are smaller in size than mouse hepatocytes were plated in 96-well plates at 0.5 × 106 viable cells per well. The plating media was Williams' Medium E (Life Technologies Cat #A12176-01) supplemented with l-glutamine (2 mM) (Life Technologies Cat #35050-061), HEPES (10 mM), insulin (10−7 M), dexamethasone (10−7 M), penicillin (100 U/mL), streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL). The same medium without FBS was used to culture attached hepatocyte monolayers for 2-3 days at 37°C in a 95% air/5% CO2 atmosphere. The medium was changed daily.Primary human hepatocytes were isolated from human tissues obtained with informed consent from each patient, according to the ethical and institutional guidelines approved by the University of Kansas Medical Center Institutional Review Board. All liver specimens were obtained in accordance with a Human Subject Committee approved protocol from patients undergoing a hepatic resection procedure or from donor organs. Primary human hepatocytes from liver specimens were isolated as previously described . BrieflyFor arsenite treatment, culture medium was replaced with fresh media containing the appropriate concentration of sodium arsenite dissolved in water and incubated at 37°C for 16 hours. All arsenite solutions were made fresh in slightly acidic (pH 6.8) Milli-Q water and immediately stored in dark filled microbottles to preserve the As (III) species .50 values were calculated based on absorbance and arsenite concentration using Sigma Plot XII.Cell viability was measured in logarithmically growing HepG2 cells as previously described . Cytotox3 Extracellular Flux Analyzer  as per manufacturer guideline. Briefly, HepG2 cells and human or mouse primary hepatocytes were seeded in XF24-V7 plates . After 6 h, 400 μL media with sodium arsenite was added to achieve a final concentration of 0, 0.3, 0.6, 1.25, 2.5, 5, and 10 μM arsenite (n = 5 wells). Cells were further incubated overnight at 37°C, 5% CO2. The XF243 sensor cartridge was hydrated with 1 mL calibration buffer per well overnight at 37°C. The sensor cartridge was loaded with oligomycin , carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone , and rotenone plus antimycin A  to measure the bioenergetics profile. Cells were washed twice with prewarmed XF assay medium containing 25 mM glucose and incubated in XF assay medium at 37°C without CO2 for 1 h. In case of mouse and human primary hepatocytes XF assay media was supplemented with 1 mM sodium pyruvate. Once the sensor cartridge was equilibrated, the calibration plate was replaced with the cell plate to measure OCR.Oxygen consumption rate (OCR) was measured in real-time, in an XF24μg of total protein was analyzed by polyacrylamide gel electrophoresis (PAGE). Primary antibodies were used to detect SOD2 , catalase , GAPDH , actin , HO-1 , and OXPHOS complexes . Immunoreactive proteins were detected using polyclonal goat anti-rabbit or anti-mouse horseradish peroxidase IgG secondary antibodies  and visualized using Supersignal™ chemiluminescent horseradish peroxidase substrate . Densitometry analysis was performed using ImageJ analysis software .For immunoblot analysis, cell lysates were prepared in 1x RIPA buffer  with protease inhibitors (Roche Applied Science). Approximately 25 μg total protein was allowed to wick on a dipstick which allowed Complex I or IV protein to be immunocaptured with anti-human or anti-mouse Complex I or IV antibody. The dipstick was then immersed in Complex I or IV activity buffer solution to form a blue-purple precipitate at the Complex I or IV antibody line on the dipstick. The signal intensity was measured by ImageJ to analyze the activity.Complex I and IV activities were measured using the Complex I Enzyme Dipstick Assay kit or Complex IV Dipstick Assay kit  as per the manufacturer protocol. Briefly, 50 6 cells were treated with different concentrations of arsenite for 16 hr. Cells were lysed with 0.5 mL of 0.5 M perchloric acid on ice for 5 min. The extraction mixture was centrifuged at 16,000 ×g for 10 min at 4°C. The supernatant was quickly neutralized to ~pH 6.0 with 4 M potassium bicarbonate. The neutralized supernatant was incubated for 30 min on ice to precipitate potassium perchlorate which was removed by centrifugation at 16,000 ×g for 10 min at 4°C. The supernatant was stored at −80°C until further analysis.A total of ×10μM–1 mM). The HPLC instrument  was equipped with an LC-20AD pump system and SPD-M20A diode array detector. Supelcosil™ LC-18 150 × 4.6 mm column (5 μm particle size) was used for separation of ATP. HPLC separation was achieved using gradient elution. Mobile phases A and B consisted of 0.1 M monopotassium phosphate and 0.008 M tetrabutylammonium hydrogen sulfate dissolved in deionized water and adjusted to pH 6.0. Two-percent acetonitrile was added to the mobile phase A while mobile phase B consisted of 30% acetonitrile. The elution program was as follows: 2.5 min 100% A, 0% B; 3.5 min 90% A, 10% B; 1 min 80% A, 20% B; 5 min 60% A, 40% B; 8 min 0% A, 100% B; and 10 min 100% A, 0% B. Flow rate of the mobile phase was 1 mL/min, while the injection volume was 50 μL. The autosampler was set at 4°C and the column was maintained at 30°C. ATP in the samples was identified by comparison with the retention time of the standard (~7 min), while the concentration was determined using a standard curve.ATP standards were made in 0.5 M perchloric acid neutralized with potassium bicarbonate as described above. ATP standards were linear for the dilution range used in this study  in phenol red free William's E medium for 15 min. Cells were then washed with Hanks Balanced Salt Solution (HBSS) and imaged on a Nikon Ti inverted fluorescence microscope with heated environmental chamber.2, 0.1 mM ethylene glycol tetraacetic acid, 0.5 mM dichlorodiphenyltrichloroethane, and 0.5% NP40). MitoSOX Red fluorescence in the cell lysate was measured at 510/580 (excitation/emission). Acetaminophen , shown previously to generate mitochondrial oxidative stress, was used as a positive control. The measured fluorescence value was expressed as a fold change compared with that of untreated control.For spectrofluorometric quantitation of mitochondrial reactive oxygen species, cells were loaded with MitoSOX Red as for imaging experiments. At the end of MitoSOX Red incubation, cells were washed with HBSS and lysed in lysis buffer  that allows growth of mouse hepatocytes in primary culture as described . Brieflyt-test and one-way ANOVA where appropriate. Statistically significant results from ANOVA analysis were computed further using the Tuckey post hoc test. All calculations were performed with SPSS statistical software package . All values are expressed as either mean ± SD or ±s.e.m.Statistical analysis of the observed values was performed using Student's μM) for 16 hours. We found that at arsenite concentrations of <10 μM primary hepatocytes showed little or no toxicity as measured by the trypan blue exclusion assay (data not shown). In general human primary hepatocytes (HPH) were more resistant to sodium arsenite treatment compared to either mouse primary hepatocytes (MPH) or human hepatoma HepG2 cells  arsenite decreased Complex I expression in a dose dependent manner in both mouse (μM might result from increased activity of Complex I at low doses (<5 μM) while high dose arsenite (>5 μM) results in decreased ETC expression and function.Previous studies have shown that the mitochondrial electron transport chain (ETC) is central to mitochondrial bioenergetics. Thus, in an attempt to define the mechanistic basis for the nonlinear effects of arsenite on mitochondrial bioenergetics, we evaluated arsenite effects on mitochondrial respiratory complex expression and activity. We found that at higher concentrations .The predominant physiological contribution of the proton circuit is the generation of ATP by oxidative phosphorylation. Thus, we tested whether changes to cellular ATP demand in response to arsenite exposure could contribute to the differential respiratory complex activity observed in hepatocytes. However, we found no significant difference in cellular ATP levels in response to arsenite exposure (Supplementary Figure  1 in Supplementary Material available online at In the absence of ATP synthesis, the proton circuit is largely completed by the proton leak. Interestingly when oligomycin was added to inhibit oxidative phosphorylation to measure coupling efficiency, primary hepatocytes demonstrated increased leak respiration independent of the arsenite concentration to which they were exposed . This suThe mitochondrial leak respiration in the presence of oligomycin is composed of proton leak that may or may not contribute to ROS generation. Thus we used live cell imaging of MitoSOX oxidation to determine if arsenite-induced mitochondrial leak respiration contributed to ROS generation in primary hepatocytes. Consistent with the increased proton leak, arsenite-induced ROS production was linear, with increasing concentration of arsenite promoting increased ROS production in a dose dependent manner in both mouse Figures  and humaμM resulted in the activation of the antioxidant stress response which reached a point of saturation beyond arsenite concentration of 2.5 μM (2O2, they were significantly more resistant to H2O2 induced toxicity compared to untreated cells (Previous studies in transformed cell lines have suggested reversal in the direction of gene expression change between low and high arsenite concentrations . These cf 2.5 μM . Consisted cells .We next tested if arsenite treatment stimulated proliferation signals in primary hepatocytes. Proliferation of mouse hepatocytes was evaluated as previously described . AlthougThe data presented support the hypothesis that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. It is to be noted, however, that the results from our studies are predominantly in vitro and that further in vivo studies are required to confirm these observations.Historically, mitochondrial ROS production and oxidative damage have been associated with toxicity. However, numerous findings have emerged in recent years indicating that oxidants in general and mitochondrial ROS in particular may act as secondary messengers in many cellular pathways, including those that promote metabolic health and longevity , 43, 44.An increasing body of evidence suggests that the site of ROS production significantly contributes to their apparent dual nature on cellular physiology. In this context mitochondrial respiratory Complex I has been shown to contribute significantly to this dual nature on cellular physiology , 45, 46.In summary, our studies suggest that in primary hepatocytes arsenite dose-response relationship, with respect to growth and survival endpoints, does not follow either the threshold or the linear characteristics but rather a U-shaped model. Further, in our model system, although the mediator of arsenite-induced biphasic response appears to be ROS, the source of such ROS appears to result from nonlinear mitochondrial bioenergetics. Evidence for As-induced hormesis has been observed with respect to three distinct measures: cell proliferation or viability, base excision DNA repair, and, recently, telomerase activity , 47, 48.Arsenic exposure did not affect cellular ATP levels significantly. suggesting that the increased oxygen consumption in response to low dose arsenite exposure was not a result of increased coupling efficiency. Interestingly, when oligomycin was added to inhibit oxidative phosphorylation to measure coupling efficneicny, primary hepatocytes demonstrated increased leak respiration suggesting increased uncoupling of respiration.


Moreover, U87MG cells, p53wt cell line was susceptible to AR antagonists in vitro and in vivo. The current study provides insight into the biological role of AR in suppressing SVIP and p53 and promoting the progression of glioma as well as the clinical treatment of glioma patients.The incidence of glioma in men is higher than that in women; however, little is known about the expression and basic function of the androgen receptor (AR) in gliomas. AR inhibited the small VCP/p97-interacting protein (SVIP) on the transcriptional level was previously reported. The present study shows that the protein level of AR is highly expressed in cell lines of the nervous system. Moreover, the AR expression is increased while SVIP expression is decreased in tumor tissue of glioma patients, which is in agreement with the progressing WHO grades. A statistically significant increase in serum testosterone level of glioma patients compared with that of non-cancer patients was also detected. Furthermore, it has been proved that SVIP is down-regulated as well as AR is up-regulated in glioma cell lines with R1881 treatment. Interestingly, the depletion of SVIP using siRNA facilitated cell proliferation and decreased p53 expression. In addition, overexpression of SVIP increased cell death only in p53 A glioma arises from glial cells. Gliomas constitute approximately 30% of all brain and central nervous system tumors and 80% of all malignant brain tumors and are rarely curable . AccordiAndrogens play a crucial role in gender differentiation, development and expression of the male phenotype, and exert their biological effects through interaction with the androgen receptor (AR) . Upon suTwo anti-androgens, ARN-509 and MDV3100 inhibiting AR nuclear translocation and AR binding to androgen response elements , 14, forIt has been reported that in response to a physiological concentration of metabolically stable androgen R1881), the small VCP/p97-interacting protein (SVIP) expression could be down-regulated by AR on a transcriptional level in prostate cancer cell line LNCaP ; however81, the sin vitro and in vivo xenograft evidence to support AR and SVIP as new targets for p53wt gliomas.Therefore, the present study aimed to demonstrate that AR signaling and serum T level are related to glioma and provided Expression of AR in 11 cell lines was analyzed by Western blot assay . The resP < 0.001) and benign brain tumor group (P < 0.001). Moreover, the serum testosterone level was remarkably higher in glioma patients of age 30, 50 years as compared to another age group (P < 0.001), irrespective of the gender. Furthermore, the serum testosterone levels were not significantly altered in different WHO grades both in male (P = 0.373) and female (P = 0.954) glioma patients, suggesting that increased serum testosterone level in glioma patients not be a result of tumor progression. Instead, the T level may rise before the tumor progress. We further analyzed the significance of serum testosterone level differences among age groups in glioma patients, benign brain tumor group, and normal control group (Table The serum testosterone (T) levels in glioma patients, benign brain tumor patients and normal controls, as well as the comparison of the serum testosterone of glioma patients among age groups and WHO grades, are shown in Table up Table . Glioma P < 0.001). Interestingly, the cells located around the blood vessels in the high-grade tumor tissues expressed AR at an extraordinarily high level , were subjected to Western blot assay and immunohistochemistry staining. It has been previously reported that SVIP is inhibited by AR . Hence, gh level . All theAR, a transcriptional regulator, was translocated in the nuclear region with R1881 treatment, as assessed by immunofluorescence in U87 cells . Unlike wt cells lines showed rapid growth with SVIP knockdown  by daily oral gavage. The mean tumor volume, which was measured with the caliper directly  is the most widely used drug for chemotherapy of glioma patients; however, in most cases, the patients and doctors are far from being satisfied with this approach, due to only slight improvement in prognosis . On the p53 mutations occur more frequently in secondary GBMs (> 60%) than in primary GBMs (~10%) and primary GBM accounts for up to 95% of the GBM cases , it can The role of AR is widely studied and recognized in prostate cancer. Thus, targeted chemotherapeutics against AR have been developed. We can also use the activity of these chemicals as a reference in the further glioma-related research based on the above theory. Not only novel targets of chemotherapy for glioma patients, but also a series of biomarkers to characterize glioma including serum testosterone, AR, SVIP, PSA, and genotype of p53 could be clinically applied in the future. However, one obstacle in glioma chemotherapy is the blood brain barrier (BBB). Only two studies reported that ARN-509 could permeabilize the BBB , 35. ThuSVIP was negatively regulated on the transcriptional level. Despite AR-p53 mutual suppression, decreased SVIP expression may lead to declined p53wt expression and facilitation of the malignancy progression that could be delayed by AR antagonists. Our results indicate that the role of AR and SVIP in the pathogenesis of different types (subtypes) of gliomas requires being defined discretely.In summary, we have found that androgens function to promote glioma cell proliferation through activating AR signaling. Then, an AR response gene The following primary antibodies were used: polyclonal anti-SVIP, monoclonal anti-NeuN (Millipore), monoclonal anti-CNS (Millipore), monoclonal anti-GFAP , and monoclonal anti-CD11b/integrin am (OX-42). Polyclonal anti-β-actin, polyclonal anti-p53, polyclonal anti-Ki67, polyclonal anti-PSA (A67-B/E3), and anti-AR (N-20) antibodies were obtained from Santa Cruz . Control siRNA and siRNA targeting human SVIP have been previously described , siRNAs 2 at 37°C. For treatment with R1881 or ARN-509 and MDV3100  both obtained from APExBio, TX, USA, cells were grown in phenol red-free DMEM (Gibco) medium supplemented with 10%  dextran-coated charcoal stripped FBS  for 72 h prior to treatment. Every 24 h, the medium was replaced with medium with or without hormone and the cells were incubated for a further 24 to 72 h before analysis. Plasmids expressing wild-type SVIP has been previously described [HeLa, SKBR-3, U251MG, and U87MG cells were obtained from China Center for Type Culture Collection . Neuro2A, SH-SY5Y, and BIU-87 cells were kind gifts from Dr. Yuxian Shen ; SW480 and SW620 cells were gifts from Dr. Siying Wang ; PC-3 and LNCaP cells were gifts from Dr. Xuemei Huang . The cells were cultured in DMEM or RPMI 1640  supplemented with 10% fetal bovine serum (Invitrogen), 100U/ml penicillin, and 100 mg/ml streptomycin in a humidified incubator with 5% COCell lysis, protein extraction, and immunoblotting were performed as described previously . The forCells or cryostat sections (5 mm thick) of rat brain tissue were fixed with 4% paraformaldehyde in PBS at 4°C for 30 min and then subjected to immunofluorescence staining as described previously . Cells owww.cellprofiler.org), and an average of the 5 view fields was presented. Soft agar assay was carried out as a classic protocol on a 6-well plate with 1000 cells/well. After 16 days, the colonies were stained with 0.005% crystal violet and observed under a microscope. Each experiment was performed in triplicate.72 h after RNA transfection or indicated treatment, the following assays were performed. For MTT assay, cells were seeded into 96-well plates at a density of 4000 cells/well. After treatment, the medium was replaced with fresh culture medium contained 0.5 mg/ml MTT reagent and incubated at 37°C for 4 h. Subsequently, the supernatant was aspirated, and cells were lysed in 200 μl DMSO for 10 min at 37°C. The optical density (OD) was measured at 490 nm using a plate reader. For cell count, the cells were split into 24-well plates. 5 images of the cells were captured on 5 random view fields from every well of a 24-well plate, by microscopy. Then, the cell numbers were estimated by Cell Profiler (6 cells/ml with cold PBS. 100 μl of the cell suspension was mixed with 10 μl of annexin V-FITC and 10 μl PI (20 μg/ml) in a tube and incubated for at least 20 min at room temperature in the dark. Subsequently, 400 μl of PBS binding buffer was added to each tube without washing and analyzed immediately by flow cytometry analysis (BD Biosciences). This assay was performed in triplicate.Apoptotic cells were differentiated from viable or necrotic cells by combined application of Annexin V-FITC and propidium iodide (PI) . The samples were washed twice and adjusted to a concentration of 1 × 10The formalin-fixed-paraffin-embedded tissue samples, including 73 glioma tissues and 12 normal brain tissues, were obtained from the patients who underwent surgical treatment from July 2011 to July 2013. Glioma tissue samples were obtained from patients with primary gliomas according to 2007 WHO classification , 8, 15, n = 71), benign meningioma (n = 83), and acoustic neuroma (n = 24). The normal control group consisted of 118 individuals with functional nervous disorders but not benign or malignant tumors and comprised of 42 males and 76 females with a median age of 51 years . Young patients (< 30 years) with functional nervous disorders were rare and were not included in the normal control group.Serum samples were collected from three groups, glioma group, benign brain tumor group, and healthy individuals. The glioma group consisted of 122 male and 67 female patients with a median age of 49 years , different from the source of tissue samples, with histologically confirmed glioma who underwent surgical treatment. The benign brain tumor group consisted of 64 male and 112 female patients with a median age of 47 years , 176 patients who underwent surgery and were histologically confirmed with pituitary adenoma  following the manufacturer's instructions. Reverse-transcriptional PCR was performed as described previously for 25 and 30 cycles in order to reduce the effect of amplification saturation [6 U87MG cells mixed with 100 μl PBS. The tumor size was measured daily with a caliper beginning 1 week after inoculation, and the volume was calculated as π/6 × length × width2. After the tumor volume had reached 100 mm3, the mouse was treated with MDV3100 (APExBio) or placebo (control) at a dose of 10 mg/kg body weight by daily oral gavage. MDV3100 stock were prepared in vehicle aqueous slurry (placebo), containing 20% PEG-400, 0.25% Tween-80, and 0.5% w/v carboxymethylcellulose (CMC) solution in 20 mM citrate buffer (pH 4.0), stored at 4°C. Xenograft tumors were excised upon sacrifice on day 14 post-drug treatment.Four weeks old male BALB/C nude mice, obtained, and maintained in specific pathogen free Animal Center of Dalian Medical University, were castrated. 1week after castration, mice were subcutaneously injected in the flank with 5 × 10In-Vivo FX Professional Imaging System equipped with fluorescent filter sets . The field of view was 120 mm in diameter. The frequency rate for NIR excitation light was 2 mW/cm2. Prior to imaging, mice were anesthetized by intra-abdominal injection of 1% pentobarbital sodium (45 mg/kg).Mice bearing human U87MG tumor xenografts were injected intravenously with IR-780 iodide (Sigma) at a dose of 0.2 mg/kg. Whole-body optical images were captured 72 h after dye injection using a Kodak The study was performed in accordance with the 1964 Helsinki Declaration. Tissue and serum sample collection were approved by the ethics committee of Anhui Provincial Hospital Affiliated to Anhui Medial University and written informed consent was obtained from all patients. All mice experiments were approved by the Dalian Medical University Animal Care and Use Committee.P < 0.05 was considered statistically significant.SPSS 17.0 was used for statistical analysis in the present study. Ranked data analysis was performed using rank-sum test. Measured data were reported as means ± SD and analyzed using one-way analysis of variance (ANOVA) with Bonferroni's post hoc analysis, respectively. Chi-squared test or Fisher's exact test was used to assess the correlation between the expression of AR and the clinicopathological features.


Extracellular miRNAs are increasingly studied as markers for specific diseases. They are released in biological fluids in a remarkably stable form, and may play a role in intercellular communication. They are thought to be protected against degradation by either encapsulation within microparticles, or by binding to proteins (mostly AGO2). The particulate forms may be internalized by endocytosis or membrane fusion, but the protein-bound forms require a receptor mechanism for their uptake. A major question is whether there are natural cell-membrane receptors that capture and internalize protein-bound functional miRNAs. We examined neuropilin-1 (NRP1), in view of its properties as a receptor for many ligands, including growth factors such as vascular endothelial growth factor (VEGF), and efficiency at mediating ligand internalization. It is expressed by endothelial cells, many other normal cell types, and cancer cells. Here, we report that NRP1 binds miRNAs with high affinity, and promotes their entry into the cell. Furthermore, the internalized miRNAs remain functional, as they specifically regulate proliferation and migration of cancer cells, as well as tube formation by human endothelial cells. Anti-NRP1 antibodies or NRP1 siRNA knockdown block miRNA effects, further confirming NRP1-mediated uptake. VEGF does not compete with miRNAs for binding to NRP1. In addition, NRP1 binds extracellular AGO2 (carrying miRNA or not), and internalizes AGO2/miRNA complexes. Because miRNA bound to AGO2 appears to the most abundant form in body fluids, this may have important physiological and pathological effects. They have also been reported to play important roles in tumor development and metastasis . ExtraceThe involvement of specific miRNAs in kidney cancer pathogenesis has been documented in the literature , 6. MoreWe hypothesized that there is a high affinity receptor for miRNAs on cells, with the capacity to internalize their ligand. In view of its properties as a multispecific receptor , and effHere we demonstrate that synthetic miRNAs bind to recombinant NRP1 with high affinity. The panel of miRNAs included miR-20a, miR-138, miR-331, miR-422, and control RNA, which were biotinylated using the Pierce biotinylation kit. The first two are reported to be deregulated in kidney cancer , 18. Bot2+ and Ca2+ altered the affinity of miRNAs, but binding occurred at physiological concentrations of these cations. Notably, addition of Mg2+ alone increased binding affinity.A typical binding curve of non-denatured miRNA binding is presented on Figure The affinity of a miRNA to NRP1 may be modulated in the presence of its other ligands, such as VEGF, also present in the circulation. We address these issues in Figure While positively-charged motifs in NRP1 are likely essential for binding of the negatively-charged miRNA, the role of other factors such as protein conformation is not clear. To examine the role of these factors, we tested the binding of miRNA to a plate coated with poly-ornithine instead of NRP1. The concentration of this positively-charged polypeptide was the same as that of sNRP1. The Kd value acquired in this experiment was 6.668±1.293. This is an order of magnitude higher than the values recorded for the binding to sNRP1 . To demonstrate translocation we conjugated the biotinylated miRNAs used in the cell-free binding assays with streptavidin-coated fluorescent microparticles, and incubated them with ACHN kidney cancer cells pre-treated or not with a blocking anti-NRP1 antibody Figure . FluoresParticle-associated fluorescence was co-localized with NRP1. We used ImageJ to quantify the colocalization. About 90% of the particle-associated fluorescence was co-localized with NRP1 in cytoplasm when the particles were conjugated with miRNA, while this value was only about 25% in the negative control and in the antibody-pretreated cells Figure . This inWe also confirmed this observation in an independent experiment. For this, we incubated NRP1-positive BT-474 breast carcinoma cells with NRP1-targeting siRNA in a serum-free medium without transfection reagent and observed the knockdown of the targeted protein Figure . The marAs noted above, the streptavidin-coated microparticles had a limited ability to bind to the cells in the absence of miRNA. To exclude the possibility that these particles directly bind to NRP1 irrespective of miRNA, we performed a cell-free binding assay Figure . It showCirculating miRNAs are found in a complex with AGO2 protein which is believed to protect them from degradation. AGO2 contains an RNA-binding domain (Piwi). We found that full-length AGO2 binds miR-20a with an affinity that is lower than that of NRP1: Kd =7.272±4.692 nM and 0.3864±0.02742 nM, respectively. Pre-treatment of miRNA with an equimolar amount of sNRP1 did not prevent the retention of miRNA by the plate-immobilized Piwi domain of AGO2 Figure . SimilarFurthermore, full-length AGO2 by itself bound to NRP1-Fc with high affinity, and miRNA neither was required for this binding nor inhibited it Figure . The truThese experiments demonstrate that NRP1 and AGO2 bind to each other irrespective of the presence of miRNA. Furthermore, it appears that NRP1/AGO2 complexes retain attached miRNA, and can internalize the bound miRNA. It is important to note that NRP1 can bind to itself, as mentioned previously, and we postulate that extracellular NRP1/AGO2/miRNA complexes have a high affinity for NRP1 on the membrane of cells.RNA internalized by a cell via an NRP1-dependent mechanism may induce specific modulation of the cell function. We studied the effect of miRNAs on the proliferation and migration of two renal clear cell carcinoma cell lines, 768-O and ACHN. Importantly, no transfection reagents were added. Three miRNAs  increased the division of 768-O cells two-to-three times compared to the negative control Figure . Pre-treIn a wound-scratch assay, miR-20a increased the migration rate of 768-O cells by 300% and miR-331 by 200%, while miR-422 was less effective Figure . MigratiRenal cell carcinoma is a vascular tumor. It was recently postulated that miRNAs released by cancer cells can be an instrument in their cross-talk with both tumor and non-tumor cells, such as vascular endothelial cells . To recoIn this study, we identify NRP1 as a high affinity receptor for extracellular miRNAs. Moreover, we show that NRP1 internalizes miRNA in either naked form, complexed to beads, or bound to AGO2. The internalized miRNAs exert their usual function.The presence of miRNA in biological fluids is well established. However, the origin of these miRNA species and their possible function are not clear. They may be released from dead cells with no specific function . They caHere, we demonstrated direct binding of synthetic miRNAs to immobilized NRP1 and calculated the Kd values. The affinity was notably increased in the presence of magnesium, and this implies that the RNA affinity to NRP1 is not solely based on the electrostatic interactions, because divalent cations neutralize the negative charge of phosphate groups. This is further confirmed by the observation that a positively-charged polypeptide, poly-ornithine, binds miRNA with a much lower affinity of Kd=6.668±1.293 nM.We observed no notable difference in retention of miR-331on the plate coated with either full-length NRP1-Fc or the truncated sNRP1 lacking the C-domain, and it appears the a1a2 and b1b2 domains are more essential for the binding than the C-domain. Furthermore, a blocking antibody against the b1b2 domains prevented miRNA binding, suggesting that the principal binding site is located in that region, but further investigation is required to confirm this. The binding curve fits a single-site model; however, this does not exclude multiple non-interacting sites with similar affinity. Retained miRNA was displaced by heparin. This raises the possibility that 20 base-long miRNAs interact with the same sites as heparin. Alternatively, heparin may induce a conformation of NRP1 not favoring the binding of RNA. The identification of binding sites for miRNA requires further studies.2+ and 1.26 mM Ca2+) and non-denatured miRNA. Thus far, all the miRNAs we have tested bind to NRP1; however, a more exhaustive study is required to identify differences in binding affinities, or miRNA species that do not bind.RNA-protein interactions have often been studied in the absence of divalent cations using denatured RNA , 22. UndThe affinity of miRNA to NRP1 may be modulated by the presence of the other ligands of this protein, such as VEGF. We found that VEGF did not notably change the affinity of miRNA to NRP1 and, similarly, miRNA did not alter the affinity of VEGF to NRP1. Recently, Fisher et al. reported that the interaction of VEGF with NRP1 was increased in a narrow range of concentration of total RNA . Their oWhile the binding of miRNAs to a recombinant protein in a cell-free assay is an important phenomenon, it is essential to show that the same interaction plays a physiological role in productive translocation of extracellular miRNA. To demonstrate binding and uptake, we conjugated biotinylated miRNA to streptavidin-coated fluorescent microparticles and incubated them with cultured NRP1-expressing cells of the ACHN renal clear cell carcinoma cell line. The treated cells were fixed in paraformaldehyde and cytoplasmic location of the particles was demonstrated by confocal microscopy. It is important to note here that unlike methanol, paraformaldehyde fixation does not induce translocation of the surface material into the cytoplasm as an artifact of fixation process.Although limited non-specific uptake of the particles was apparent, the number of the particles bound and internalized by the cells was dramatically increased by their conjugation with biotinylated miRNA. In a cell-free assay, the beads without miRNA did not bind to NRP1 more than to a BSA-coated control plate, but binding to NRP1 was increased in the presence of biotin-conjugated miRNA. Particle-labeled miRNA co-localized with NRP1 both on the cell surface and in the cytoplasm. Indeed, more than 85% of the particle-associated green fluorescence was co-localized with NRP1-associated fluorescence signal. NRP1-blocking antibody eliminated the miRNA-mediated increase the green fluorescence in cytoplasm. A small fraction of ACHN cells was naturally NRP1-negative, and they did not uptake the particles. Moreover, siRNA knockdown of NRP1 greatly reduced bead uptake. Therefore, NRP1 binds extracellular miRNA and assists its translocation across the cell membrane. The intracellular fraction of miRNA was notably high compared to the surface-bound fraction.The remarkable stability of miRNA in the circulation is largely attributed to complex formation with the RNA-binding protein AGO2 . This coAlthough translocated into the cytoplasm, miRNA may remain nonfunctional, if it is degraded, trapped in an intracellular organelle, or masked by a protein. To demonstrate productive translocation, we studied the proliferation and migration of 768-O and ACHN cells, treated or not with various miRNA mimics from our panel. In NRP1-expressing cells, but not cells subject to siRNA knockdown of NRP1, unassisted miRNA uptake significantly modulated proliferation. This occurred in a miRNA species-dependent and cell-dependent way. Similar results were obtained in a cell migration assay. For example, treatment with miR-20a increased migration, and this effect was neutralized by a competing miRNA that does not affect migration by itself. Importantly, in the presence of NRP1, but not when it was blocked by antibody or knocked down by siRNA, the miRNAs exerted their expected effects by increasing the rate of migration. Taken together, these findings suggest that the observed effects on proliferation and migration are the result of canonical miRNA signaling. Although most of the cell-bound miRNA is found in cytoplasm, a small fraction of it is membrane-bound, and theoretically it can act in a noncanonical way. This requires further investigation.Because NRP1 interacts with several growth factors and other ligands, its blockade can alter responses in tumour-cell assays. However, our results with siRNA knockdown strongly support productive internalization of the miRNAs. With this approach, NRP1-low cells are tested with our without miRNA, and can be compared to NRP1-high cells irrespective of other factors. Our results show that NRP1-low cells did not respond to miRNAs. Moreover, in the bioassays we report, the NRP1 blocking antibody effect (without miRNA) was modest and insufficient to alter the interpretation of the results. These findings demonstrate that NRP1 was required to internalize the miRNAs such that they exerted their functions.Although identification of the miRNA targets is beyond the scope of the current study, our observations match the published data by others. Thus, miR-422 was associated with relapse-free survival of the patients with hepatocellular carcinoma  and concThe mechanisms by which NRP1 induces endocytosis of ligands have not been completely elucidated, but two potential pathways have been described. NRP1 has a cytoplasmic C-terminal motif (SEA) that binds to the PDZ protein synectin  , 29. It In conclusion, NRP1 binds miRNA with high affinity, and is able to translocate its cargo across the cell membrane. Furthermore, AGO2/miRNA complexes are also bound and internalized. The internalized miRNAs preserve their function (productive transfer). Signaling through this mechanism is limited to NRP1-positive cells. It is possible that some selectivity occurs because miRNA species vary in their affinity for the receptor, and this warrants further investigation. Moreover, the response is likely to be cell-type dependent. NRP1 is expressed by many cell types, and we postulate NRP1-dependent internalization of miRNAs has an important physiological function.R streptavidin coated fluorescent microparticles labeled with Dragon Green (mean diameter 0.22 μm) were from Bangs Laboratories, Inc. . Anti-NRP-1 antibody  home-labeled with AlexaFluor 647 was used for flow cytometry analysis and cell imaging. Additionally, to block NRP1--dependent responses, we used monoclonal antibody MNRP1685A, which is an anti-human/murine NRP1 obtained from Genentech, which binds to the b1b2 domain of NRP1. Phalloidin labeled with AlexaFluor 568 (Molecular Probes (Thermo Fisher Scientific), and DAPI were used for counter-staining in the internalization experiments. CytoFix-CytoPerm kit from BD Biosciences  was used to fix and permeabilized cells. Cell Trace CFSE cell proliferation kit for flow cytometry was from Life Technologies. Matrigel matrix basal membrane growth factor-reduced was from Corning . NRP1-targeting and scrambled siRNA were from Santa Cruz Biotechnology. Lipofectamin 3000 (Life Technologies) was used for the knockdown, as indicated. Human recombinant AGO2 (Piwi domain and a full-length protein) was from Biorbyt and anti-pan-AGO2 antibody was from EMD miRNA mimics were purchased from Life Technologies (Thermo Fisher Scientific). Recombinant full-length rat neuropilin-1/Fc chimera (NRP1-Fc), truncated human neuropilin-1 , IgG-Fc portion, and VEGF165 were from R&D Systems (Minneapolis). NRP1-Fc has unmodified extracellular domain, while transmembrane and cytosolic domains are replaced with a C-terminal portion of human IgG-Fc. Nuclease-free BSA was from the New England Biolabs . Nuclease-free water was used for solutions . NUNC ELISA plates (Thermo Scientific) and Luminata™ Forte ELISA HRP Substrate ) were used for the luminometric binding assay. ProActiveIt was performed using Pierce™ RNA 3′ biotinylation kit from Thermo Scientific, according to the manufacturer's protocol. It is based on the ligation of biotinylated cytidine (bis)phosphate to the 3′-end of oligonucleotides. This modification is not expected to affect the binding affinity of miRNA, because it leaves the 5′-end and the seed unmodified. Non-labeled RNA control from the kit(5′-CCUGGUUUUUAAGGAGUGUCGCCAGAGUGCCGCGAAUGAAAAA-3′) was labeled along with the miRNA species and tested in the binding assay or used as an unlabeled competitor RNA. Denaturation of miRNA was performed as described in the same protocol.2 or sterile nuclease-free HBSS with calcium (1.26 mM) and magnesium (0.9 mM). The plate was incubated with streptavidin-peroxidase (R&D Systems) for 20 min. After the wash the plate was kept in the dark for 20 min before the substrate was added in the dark room to minimize auto luminescence. The plate was read using a SpectraMax 5M luminometer-plate reader. The signal integration time was 500 ms. The signal was stable within at least 10 min. Specific binding was calculated by subtraction of the values for the non-specific binding from total binding .ELISA plates were coated with NRP1-Fc, sNRP1, AGO2, or with IgG-Fc and blocked with nuclease-free BSA. Non-coated wells treated with BSA were used to estimate the non-specific binding. 0.05% TWEEN in nuclease-free water was used as a wash buffer. Biotinylated miRNA was either denatured at 85°C for 8 min or used undenatured. Binding of miRNA was performed either at 4°C overnight or for 2 h at 37°C. The binding buffer was sterile PBS with or without 0.5 mM MgClTo examine whether fluorescent streptavidin-coated microbeads used in some experiments had affinity for NRP1-Fc or NRP-Fc/miRNA, plates were coated with NRP1-Fc, or BSA alone, as described above. These plates were incubated, or not, with biotin-conjugated miRNA, and then incubated with the fluorescent streptavidin-coated microbeads with shaking for 20 min. In this case, the beads were resuspended in 0.05 % TWEEN in PBS at 1:1000 ratio and added to the black ELISA plate containing immobilized proteins, with or without retained biotinylated miRNA. The fluorescence was read using ELISA reader with 480 nm excitation and 520 nm emission wavelengths.To study the effect of VEGF on the binding of miRNA, the wells coated with sNRP1 and blocked were pre-treated with 1 nM recombinant VEGF for 1 h at room temperature. miRNA was added after wash-out of the unbound VEGF and incubated for 2 h at 37°C. We tested the effect of AGO2 on the miRNA retention by NRP1 and the effect of NRP1 on the miRNA binding to AGO2 in a similar way. Equimolar mix of AGO2 and miRNA in Ca/Mg-HBSS was incubated for 1 h at 37°C and diluted serially for the binding assay. The detection of the bound miRNA was performed as above.To study the effect of miRNA on the binding of VEGF a plate was coated with sNRP, blocked, and pre-treated with miRNA for 2 h before adding VEGF. The bound VEGF was detected with anti-VEGF primary antibody (R&D Systems) and secondary anti-mouse IgG-HRP (Promega) with TMB substrate. Binding of AGO2 to NRP1-Fc was studied in a similar way. In addition, equimolar mix of AGO2 and miRNA in Ca/Mg-HBSS was incubated for 1 h at 37°C and diluted serially for the binding assay to study the binding of the AGO2-miRNA protein complex to NRP1. Protein retention was quantified using anti-pan AGO2 primary antibody (EMD) and secondary anti-mouse IgG-HRP  with TMB substrate. The binding was expressed in arbitrary units defined as OD450, after the subtraction of the non-specific binding.4 cells were resuspended in serum-free medium containing 1 mg/ml RNAse-free BSA and incubated with 5 pmol miRNA in a total volume of 300 μL for 30 min at 37°C with periodic gentle mixing. After the incubation they were plated to be used in the proliferation or wound-scratch assays.Renal Clear Cell Carcinoma cells 768-O and ACHN were grown in RPMI-1640 supplemented with 10 % FBS. HUVEC cells were grown in F12K supplemented with ECGs , heparin (0.1 mg/ml) and 10 % FBS. BT-474 cells were grown in DMEM, supplemented with 10% FBS. For loading with miRNA cells were harvested with Ca/Mg-free HBSS+5 mM EDTA. 1.5×104 cells per well. Before the assay the cells were rinsed with the serum-free medium and pre-treated or not with blocking anti-NRP1 antibodies (20 μg/ml each) for 30 min in the incubator. In some cases miRNA was pre-treated with 50 nM of either sNRP1 or recombinant AGO2 (as indicated in the legends). For the conjugation, 5 pmol of biotinylated miRNA were mixed with 1-10 μl of the fluorescent streptavididin-coated microparticles and 1 mg/ml BSA in total volume of 20 μL. The mixture was vortexed for 15 min at room temperature and low speed, protected from light, as recommended by the manufacturer of the beads. After the conjugation step miRNA was diluted with serum-free BSA-containing medium (1 mg/ml) to the final concentration of 16 pM and loaded onto the pre-treated cells for 30 min at 37°C. After the incubation the cells were rinsed in serum-free medium, fixed in paraformaldehyde, permeabilized, and counter-stained with anti-NRP1-AlexaFluor 647, phalloidin-AlexaFluor 568, and DAPI. The slide was mounted in ProLong Gold (Molecular Probes) and analyzed using Zeiss LSM700 confocal microscope with 63x magnification and z-sections with 1μm interval. Co-localization of the microparticles and NRP1 was estimated using ImageJ. RNA uptake was estimated as an average fluorescence intensity of the fluorescent beads per cell area delineated by F-actin staining, in z-stacks.ACHN cells were seeded onto the chamber-slide at 2×105 cells were incubated with 60 pmol of siRNA in serum-free medium for 30 min at 37°C.Unassisted translocation of siRNAs was demonstrated in the NRP1-expressing BT-474 cells as following: NRP1-targeting siRNA or scrambled siRNA (Santa Cruz Biotechnology) was added to the cultured cells in serum-free medium for 4 h before replacing it with DMEM, containing 10 % FBS. 72 h later the cells were harvested, fixed, permeabilized, and stained against NRP1. Total expression of this protein was evaluated by flow cytometry. Similarly, knockdown of NRP1 in 786-O and ACHN cells was achieved by the unassisted uptake of the siRNA, and the 10In the experiments of renal cell carcinoma proliferation and migration only, for maximal knockdown of NRP1, we used Lipofectamin 3000 and the same amount of siRNA as for the unassisted transfection, and a protocol described by the manufacturer.6 cells were mixed with 1.2 μl CFSE in 1 ml of the same medium and incubated for 20 min at 37°C. After rinsing in RPMI-10 % FBS and serum-free RPMI, 3×104 CFSE-labeled cells and 10 pmol miRNA mimic were resuspended in 600 μl of the serum-free medium for 30 min at 37°C. After the incubation and loading with miRNA , with or without the pre-treatment with the NRP1-blocking antibodies, they were transferred into the complete growth medium and plated. They were analyzed by flow cytometry after 24 h or 48 h in culture and staining with propidium iodide. Proliferation was quantified as a % divided cells in FCS Express 4.Renal Clear Cell carcinoma 768-O cells were harvested with HBSS-EDTA, rinsed in serum-free RPMI 1640. 10ACHN and 768-O cells were harvested with HBSS-EDTA, pre-treated or not with the anti-NRP1 antibody or a competitor miRNA (as indicated), loaded with miRNA mimics in serum-free medium as above and grown to 80% confluence in 24 well plates. The competitor RNA was used in the same concentration as the tested miRNA and was pre-loaded 30 min before miRNA was added. The wound was scratched with a 200-μl tip, and the area covered by migrating cells was calculated at various time points using ImageJ.96-well plate was coated with 50 μl Matrigel diluted with unsupplemented F12K medium 1:1. HUVEC (passages from 6 to 12) were pre-treated or not with NRP1-blocking antibodies and loaded with miRNA as described above. They were plated onto the Matrigel-coated plate at 15000 cell per well. The cells were photographed after 2, 6, and 20 h. The total length of the formed tubes was calculated in ImageJ.Statistical analysis was performed in Prism 6 GraphPad Software using ANOVA. P value ≤0.05 was considered statistically significant.


Synechococcus elongatus PCC 7942 has indeed a large capacity of FFA production, which is comparable to that of triacylglycerol production in green algae, but the yield of secreted FFAs is low because the cells accumulate most of the FFAs intracellularly and eventually die of their toxicity. To increase the FFA productivity, enhancement of FFA secretion is required.Cyanobacterial mutants engineered for production of free fatty acids (FFAs) secrete the products to the medium and hence are thought to be useful for biofuel production. The dAS1T mutant constructed from −1 of palmitic acid. This suggested that when FFA accumulates in the medium, it would inhibit the release of FFA from the cell, leading to FFA accumulation in the cell to a toxic level. To remove FFAs from the medium during cultivation, an aqueous-organic two-phase culture system was developed. When the dAS1T culture was overlaid with isopropyl myristate (IM), the final cell density, cellular chlorophyll content, and the photosynthetic yield of PSII were greatly improved. The total amount of extracellular FFA was more than three times larger than that in the control culture grown without IM, with most of the secreted FFAs being recovered in the IM layer. The cellular FFA content was decreased by more than 85% by the presence of the IM layer. Thus, the two-phase culture system effectively facilitated FFA secretion out of the cell. An average FFA excretion rate of 1.5 mg L−1 h−1 was attained in the 432 h of cultivation, with a total amount of excreted FFA being 0.64 g L−1 of culture. These figures were more than three times higher than those reported previously for the cyanobacteria-based FFA production systems.Growth of dAS1T cells but not WT cells was inhibited in a liquid medium supplemented with 0.13 g LRemoval of FFA from the culture medium is important for improving the productivity of the FFA production system using cyanobacteria. Further increase in productivity would require an increase in both the rates of FFA production in the cell and active FFA export across the plasma membrane.The online version of this article (doi:10.1186/s13068-017-0831-z) contains supplementary material, which is available to authorized users. Photosynthetic microorganisms including cyanobacteria and eukaryotic algae appear to be promising hosts for sustainable biofuel production because they have a high capacity for oxygenic photosynthesis , 2. Actuaas gene coding for acyl-ACP synthetase, which activates FFAs via esterification to acyl carrier protein (ACP), is inactivated to prevent the recycling of the FFAs released from membrane lipids [Escherichia coli (‘tesA) [Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942, and Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803 has been the most successful [−1 h−1 for 450 h [Chlorella vulgaris and Nannochloropsis gaditana, ranging from 2 to 3 mg L−1 h−1 [Unlike TAG production using green algae, production of free fatty acids (FFA) using genetically engineered cyanobacteria results in secretion of the product into the medium –10. If te lipids , 12. Sec (‘tesA)  is intro (‘tesA) . Among t L−1 h−1 , 15.S. elongatus PCC 7942 were less active in FFA secretion than the SD277 mutant of Synechocystis sp. PCC 6803 [S. elongatus PCC 7942 simply by inactivation of aas and introduction of ‘tesA, has a much larger capacity to produce fatty acids; it turned out that unlike the SD277 strain that secrete ~90% of FFA out of the cell [−1 h−1, while secreting FFA at a rate of 0.35 mg L−1 h−1 [S. elongatus [Although all the FFA-producing strains constructed from PCC 6803 , 16, KatIn this study, we developed an aqueous-organic two-phase culture system to remove FFAs from the medium during cultivation of dAS1T. The rate of FFA secretion and the total amount of secreted FFA in the two-phase system were more than three times higher than those reported for the SD277 strain, verifying the importance of removal of FFAs from the culture medium in the enhancement of FFA production. Effects of the two-phase culture system on growth and FFA productivity of the dAS1T strain are described and the strategy for further improvement of FFA production is discussed.−1) of palmitic acid (PA) on growth of the wild-type (WT) and the FFA-producing strain (dAS1T) of S. elongatus PCC 7942. PA is the major FFA species secreted by dAS1T and the concentration of PA tested in Fig. Synechocystis sp. PCC 6803 was reported to attain an extracellular FFA concentration of 0.20 g L−1 [Figure E. coli and Saccharomyces cerevisiae strains [S. elongatus PCC 7942 were examined. Growth of WT cells was completely inhibited when the culture was overlaid with De, Un, or TBP, but not with IM  family. Targeted disruption of one of these (rndA1–rndB1) rendered the cells highly sensitive to FFAs, indicating its involvement in FFA export [The FFA-producing strains constructed from the cell . We prevA export . PassiveA export . HoweverA export , 21. By −1 h−1 , was used as WT in this study [aas-deficient mutant expressing a foreign thioesterase gene from E. coli (‘tesA) transcriptionally fused to the nirA promoter of S. elongatus PCC7942 [3 as a nitrogen source and when appropriate, supplemented with 0.1% ethanol and 0.13 g L−1 of palmitic acid (PA). WT and dAS1T cells were grown at 30 °C under high-light conditions (180 µE m−2 s−1) with aeration with 2% (vol/vol) CO2. In all the experiments, cells precultured under low-light conditions (50 µE m−2 s−1) to the exponential phase of growth (OD730 = 0.5–1.5) were used for inoculation of fresh medium, because the PSII activity of dAS1T sharply declines during the stationary phase of growth under the high-light conditions [A derivative of is study . The FFA PCC7942 . The bas PCC7942 . The mednditions . For two730) of the cultures was determined using a Shimadzu UV1700 spectrophotometer. Dry weight of cyanobacterial biomass in liquid cultures was estimated from OD730 of the cultures  in a unit volume of culture, 10 mL aliquots of the cultures of various cell density (OD730 = 2.0–13.8) were vacuum-filtered through a pre-weighed MF-Millipore™ Membrane Filter . The cells on the filter were washed three times with 10 mL of deionized water and then dried at 75 °C until constant weight was reached. When DCW of the cultures overlaid with IM was measured, cells from 10 mL aliquots of the cultures were collected by centrifugation at 1700g for 10 min and washed three times with 10 mL of deionized water by resuspension and recentrifugation before vacuum filtration so that the effects of contaminating IM is minimized. For estimation of the amount of IM absorbed to the membrane filter, the BG-11 medium was overlaid with IM without cyanobacterial cells and incubated just as the cyanobacterial cultures. The IM-treated medium was passed through a pre-weighted membrane filter, and the filter was washed and dried for weighing. Twenty-six sets of data obtained with independent cultures were fitted to a linear relationship  .Chlorophyll was determined as described by Mackinney  and the −1.The concentration of FFA in the culture medium (or the aqueous layer in the two-phase cultivation system) and the cellular FFA content were determined as described by Kato et al. . The conAdditional file 1: Figure S1. The relationship between OD730 and the dry cell matter content of the cyanobacterial cultures. Dry cell weight (DCW) per L of cyanobacterial cultures (Y-axis) and optical density at 730 nm (OD730) of the cultures (X-axis) were determined as described in “y = 0.255x − 0.144 (r2 = 0.942). Data shown are from 26 samples of S. elongatus PCC7942 cultures.Additional file 2: Figure S2. Effects of IM on determination of FFA using the Free Fatty Acid Quantification Kit (BioVision). Fresh IM (circles) and the IM layer incubated for 240 h with the culture of dAS1T (triangles) were mixed with Assay Buffer in the kit at various ratios to give a total volume of 50 µL and then analyzed according to the manufacturer’s instruction.Additional file 3: Figure S3. A standard curve for FFAs dissolved in isopropyl myristate. A concentration series of palmitic acid (PA) dissolved in isopropyl myristate (IM)  was analyzed using the Free Fatty Acid Quantification Kit (BioVision) as described in the text. One of the essentially same results obtained in more than ten independent measurements is shown.