Benzamide

Reagents. All the chemical reagents unless otherwise stated were obtained from Sigma-Aldrich (Hamburg, Germany).
Cell culture and generation of stable cell lines. Human embryonic kidney (HEK293T) and breast adenocarcinoma (MDA-MB-231 and MCF7) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (PAA, Pasching, Austria) or DMEM/Ham F-12 medium (PAA) (mixed 1:1) with 10% fetal calf serum (Gibco, Grand island, NY) and 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml amphotericin (Sigma-Aldrich) in a humidified 5% CO₂/95% air incubator at 37 °C. For stable transfection, MDA-MB-231 cells were seeded at 2 × 10⁶ cells/plate in P100 plate and after 16–24 hours transfected with 10 µg/plate of Myc-pcDNA3, Myc-pcDNA3-MNK1a, or Myc-pcDNA3-MNK1b using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. After 24 hours, medium was changed with new medium containing 1 mg/ml of Geneticin (Invitrogen). Untransfected MDA-MB-231 cells were treated in parallel to check geneticin-induced cell death. After several passages, the presence of MNK1a or MNK1b were checked by immunocytochemistry, western blot, and mRNA quantitation, and the cells were frozen in 10% dimethyl sulfoxide /fetal bovine serum in liquid nitrogen until use (Supplementary Figure S2).
Protein extraction, dodecyl sulphate-polyacrylamide gel electrophoresis, and immunoblotting. To obtain cell lysates, cells were mechanically harvested and washed once with cold buffer A (20 mmol/l Tris–HCl pH 7.6, 1 mmol/l dithiothreitol (DTT), 1 mmol/l ethylenediaminetetraacetic acid, 1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l benzamidine, 10 mmol/l sodium molybdate, 10 mmol/l sodium β-glycerophosphate, 1 mmol/l sodium orthovanadate, 120 mmol/l potassium chloride (KCl), 10 µg/ml antipain, 1 µg/ml pepstatin A, and leupeptin). Next, we lysed the cells in the same buffer containing 1% Triton X-100 (volume ratio 1:2) and centrifuged at 12,000g for 10 minutes. Afterwards, we determined the protein concentration by the method of Bradford,^{38 (link)} and the supernatant was aliquoted and stored at −80 °C until use.
Proteins were resolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, at the conditions indicated in figure legends, and transferred onto polyvinylidene difluoride membranes. Membranes were incubated with monoclonal antibodies for 2 hours at room temperature and with polyclonal antibodies overnight at 4 °C. After washed, membranes were incubated with the corresponding peroxidase-conjugated secondary antibody for 1 hour at room temperature, developed with enhanced chemiluminescence's kits (GE Healthcare, Barcelona, Spain). Full Range Rainbow molecular weight markers (GE Healthcare) were used in all the experiments. The blots were probed with anti-eIF4E (ser209P; Cell Signaling, Danvers, MA), anti-eIF4E (BD Biosciences, Franklin Lake, NJ), anti-MNK1 (C-20), MNK2 and c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin (Sigma-Aldrich) antibodies.
Expression and purification of recombinant proteins. Recombinant HIS-MNK1b (rMNK1b) was cloned in the pDest expression vector, and the recombinant protein was purified by affinity chromatography on Ni-NTA resin columns as described.^{39 (link)} Briefly, the BL21 cells expressing rMNK1b were harvested and suspended in sonication buffer (20 mmol/l Tris–HCl pH 7.8, 0.5 mol/l NaCl, 20 mmol/l imidazole, 2 mmol/l MgCl₂, 1 mg/ml lysozime) and incubated in ice for 30 minutes. Afterwards bacteria were subjected to sonication for four cycles of 15 seconds at maximum amplitude and centrifuged at 10000g for 30 minutes at 4 °C. rMNK1b present in the soluble fraction was purified using the Histrap FF column (GE Healthcare) with an AKTA prime system plus (GE Healthcare) by elution at 0.5 mol/l imidazole. Purified HIS-MNK1b was dialyzed against phosphate-buffered saline (PBS), concentrated using Amicon columns (Millipore, Darmstadt, Germany) aliquoted and frozen at −80 °C. Protein concentration was determined as above.
Human GST-tagged MNK1a, MNK1b, and the truncated forms MNK1aΔ77 and MNK1ΔCt were subcloned into the BamHI and NotI sites of pGEX-4T3 and expressed in Escherichia coli Rosetta cells. The proteins were purified with glutathione–Sepharose (GenScript, Piscataway, NJ) according to the manufacturer's instructions. Briefly, expression was induced with 1 mmol/l isopropyl β-D-1-thiogalactopyranoside for 2 hours at 25 °C. The cells were suspended in buffer containing 5 mmol/l sodium phosphate, 150 mmol/l NaCl, 1 mmol/l ethylenediaminetetraacetic acid, pH 7.4, and 1 mg/ml lysozyme and incubated in ice for 30 minutes. Afterwards, 0.5% Triton X-100 was added, and bacteria were subjected to sonication. After removal of cell debris by centrifugation, the supernatant was incubated with glutathione–Sepharose equilibrated in the same buffer by rocking for 2 hours at 4 °C. After extensive washes with the same buffer, the proteins were eluted with 10 mmol/l glutathione in 50 mmol/l Tris/HCl at pH 8.
In vitro selection. Selection of DNA aptamers for recombinant rMNK1b was performed as described previously by Ramos et al.^{40 (link)} Briefly, synthetic random ssDNA (IBA Life Sciences, Goettingen, Germany), containing a central randomized region of 40 nucleotides flanked by two conserved 18-nucleotides regions in each end (RND40, 5′-GCGGATGAAGACTGGTCT-40N-GTTGCTCGTATTTAGGGC-3′), was denatured at 90 °C for 10 minutes and then cooled on ice for 10 minutes. For the initial SELEX round, 50 µg (2 nmol) of RND40 were mixed with 4 μg (100 pmol) of rMNK1b in 200 μl of selection buffer (20 mmol/l Tris–HCl pH 7.4, 1 mmol/l MgCl₂, 150 mmol/l NaCl, and 5 mmol/l KCl) and incubated at 37 °C for 1 hour. The aptamer–rMNK1b complexes were purified by adding 20 μl of Ni-NTA superflow (Qiagen, Madrid, Spain) for 1 hour at 4 °C. After washing three times with 1 ml of selection buffer, the aptamer–rMNK1b complexes were suspended in 20 μl of distilled H₂O and amplified by PCR using the primers named F3 (5′ GCGGATGAAGACTGGTGT 3′) and R3 (5′ GTTGCTCGTATTTAGGGC 3′) (IBA Life Sciences) under the conditions of 0.8 μmol/l/primer, 200 μmol/l dNTPs, 2 mmol/l MgCl₂, and 2 U Taq polymerase (Biotools, Madrid, Spain) in a final volume of 50 μl for 15 cycles (95 °C for 30 seconds, 56 °C for 30 seconds, and 72 °C for 30 seconds), and PCR product was ethanol-precipitated. In the next rounds of selection, 25 µg (1 nmol) of previously selected population were denatured at 90 °C for 10 minutes and then cooled on ice for 10 minutes and used as above. In addition, after round 5, the incubation time was reduced to 30 minutes. Contraselection against Ni-NTA resin was performed after rounds 4, 7, and 10.
Enzyme-linked oligonucleotide assay. To assess the enrichment of the selected population and the affinity of the individual aptamers for the target, we performed ELONA assays in which aptamers were labeled by PCR using 5′ digoxigenin-labeled F3/5′ phosphate-labeled or 5′ phosphate-labeled F3/5′ digoxigenin-labeled R3 primers (IBA Life Sciences) and removing the phosphate-labeled strand with 1 U λ-exonuclease (New England Biolabs, Ipswich, MA) during 30 minutes at 37 °C. Alternatively, we use digoxigenin or biotin-labeled ssDNA aptamers provided by IBA Life Sciences. Recombinant proteins were diluted to 1 µg/ml in selection buffer, and 200 μl of the solution were incubated in a 96-well microtiter plate (NUNC, Rochester, NY) overnight at 4 °C and, then, washed four times in selection buffer. Afterwards, digoxigenin-labeled aptamers or digoxigenin-labeled RND40 library were diluted in selection buffer at concentrations indicated in the figures, denatured for 10 minutes at 95 °C and cooled for 10 minutes on ice. Next, 200 µl of the solution were added to each well, the plate incubated at 37 °C for 1 hour, and then washed four times with selection buffer to remove unbound ssDNA. Afterwards, 200 μl of a 1/1,000 dilution of anti-digoxigenin antibody (Roche, Basel, Switzerland) or streptavidin (GE Healthcare) conjugated with horseradish peroxidase were added to the individual wells. Following 1-hour incubation at 37 °C on a shaking platform, the plates were washed four times and developed using ABTS solution (Roche) according to the manufacturer's instruction. OD_405nm values were determined using a SpectraFluor microplate reader (TECAN, Barcelona, Spain).
Analysis of aptamer-MNK1 complexes by real-time PCR. An alternative method to determine the affinity of aptamers for MNK1b is quantifying aptamers capable to bind to resin–MNK1b complex by qPCR. The complexes were obtained by incubating His-MNK1b with Ni-NTA agarose resin for 1 hour at 4 °C on a shaker. The individual aptamers (2 µg; 80 pmol) were incubated with 10 µl resin–MNK1b complexes (200 ng/tube, 5 pmol/tube) for 30 minutes at 37 °C with stirring. In parallel, the same amount of each aptamer was incubated with 10 µl of Ni-NTA resin without MNK1b. After centrifugation at 12,000g for 10 minutes, complexes were washed three times with 250 µl of selection buffer, and finally, the resin was suspended in 20 µl of H₂O and incubated at 90 °C for 10 minutes. Quantitative analysis was performed by qPCR using SYBR Premix Ex kit TaqTM (Takara Bio, Shiga, Japan) and F3 and R3 oligonucleotides following the manufacturer's instructions in a iQ5 equipment (Bio-Rad, Barcelona, Spain). The reaction mixture consisted of 1× SYBR Premix Ex Taq, 0.2 µmol/l oligonucleotide, and 1 µl of template in a 20 µl/tube final volume.
Aptamer cloning and sequencing and secondary structure prediction. The dsDNA products with “A”-overhangs from SEL7MNK1b or SEL10MNK1b were cloned onto pGEM-T Easy-cloning vector (Invitrogen) following manufacturer's instructions. Individual clones were sequenced using T7 (5′-TAATACGACTCACTATAGGG-3′) and Sp6 primers (5′- ATTTAGGTGACACTATAGAA-3′) (IBA Life Sciences). Selected ssDNA molecules were subjected to secondary structure prediction using the mFold software (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form)^{41 (link)} at 37 °C in 150 mmol/l [Na⁺] and 1 mmol/l [Mg⁺²] and QGRS Mapper, a web-based server for predicting G-quadruplexes in nucleotide sequence.^{42 (link)}Aptamer stability assays. Three-hundred nanograms of aptamer were incubated with 0.1 U DNAse I for 2 hours, and samples were collected at the times indicated in the figures using phenol-chloroform extraction. The digested aptamer was ran on a 3% concentration MS-8 Agarose gel (Conda, Madrid, Spain) in 1× TAE buffer and visualized with GelRed (Biotium, Hayward, CA).
In vitro kinase assay. In vitro MNK1a activity was assayed as follows: 0.25 μg recombinant activated MNK1a (GST-MNK1a T385D) (BPS Bioscience, EEUU) was preincubated with 0.2 μmol/l of each aptamers (6 pmol/tube) or the same concentration of the MNK1 inhibitor (CGP57380) for 5 minutes at 30 °C, and the kinase reaction was performed using as substrate 200 μmol/l of the peptide substrate (RRRLSSLRA) in 20 mmol/l Tris–HCl, pH 7.5; 50 mmol/l KCl, 10 mmol/l MgCl₂, 100 μmol/l ATP, and 1 μCi [γ -³²P] ATP (Hartmann Analytic, Germany) for 30 minutes at 30 °C. Reactions were stopped onto Whatman P81 filters (GE Healthcare), filters washed three times in 1% phosphoric acid for 10 minutes, and the radioactivity read on a scintillation counter.
For in vitro MNK1b activity assay, Myc-MNK1b were purified from transfected HEK293T cell lysates (1.5–2 mg of protein) by immunoprecipitation. Briefly, Myc antibodies were added for 2 hours at 4 °C, and complexes were collected on protein G-agarose for 1 hour at 4 °C. The beads were washed three times with 1 ml of buffer A each, once with 0.5 mol/l LiCl and twice with kinase buffer (20 mmol/l Tris–HCl, pH 7.5; 50 mmol/l KCl, 10 mmol/l MgCl₂). From the last washing, beads (1/10 volume) were transferred at new tubes, and kinase reactions were performed as above.
Translation assays. The assay was performed with the Rabbit Reticulocyte Lysate System, Nuclease Treated (Promega, Madrid, Spain). The aptamers were heated at 95 °C for 10 minutes in selection buffer and cooled on ice prior to addition. The reaction (25 µl) was performed following kit instructions, with 0.25 µl of luciferase RNA control (provided in the kit) and the aptamers at a final concentration of 4 µmol/l. The reaction was stopped at different incubation times (with emetine), and 2 µl of sample were used for measurement of luciferase activity with luciferase assay reagent (Promega) in a luminometer (Berthold, Bad Wildbad, Germany).
Aptacytochemistry. Aptacytochemistry is a technique that allows the microscopic localization of proteins present in the cells using aptamers. For this assay, stable MDA-MB-231 cells expressing Myc-MNK1a or Myc-MNK1b (4 × 10⁴ cells/well) were seed on glass coverslips pretreated with poly-l-lysine (Sigma-Aldrich). After 16–24 hours, the cells were fixed with cold methanol for 20 minutes at −20 °C, washed three times with PBS, and blocked with 10% fetal bovine serum diluted in PBS (blocking buffer) for 1 hour at room temperature. Next, the cells were incubated with 2 pmol of 5′ Alexa 488-conjugated aptamer (IBA Life Sciences) in selection buffer with 0.2% bovine serum albumin for 1 hour at room temperature. Subsequently, cells were washed three times with PBS and incubated with anti-MNK1 (C-20) (1/50 dilution) or anti-MNK1 (M-20) (1/25 dilution) antibodies in blocking buffer overnight at 4 °C. After incubation, cells were washed with PBS and incubated with rhodamine-conjugated goat antibody (Jackson ImmunoResearch Laboratories, Suffolk, UK; 1/200 dilution) in blocking buffer for 1 hour at room temperature. In other assay, MDA-MB-231 cells transiently transfected with pcDNA3-Flag-MNK1b were incubated with mouse anti-Flag antibody (Sigma-Aldrich; 1/3,000 dilution) and mouse Alexa 568-conjugated IgG (Invitrogen; 1/300 dilution) as secondary antibody. Finally, the cells were mounted on glass slides using glycerol-buffer containing p-phenyl-enediamine and 30 µmol/l bis-benzamide (Hoechst 33342) for nuclear staining. Controls were made by omitting the primary antibody. Co-localization was assessed by confocal microcopy using a Nikon ECLIPSE Ti-e inverted fluorescence microscope equipped with a Nikon C1 laser scanning confocal microscope system (Nikon, Tokyo, Japan) and a 60× oil immersion objective.
Cell viability (MTT) assays. HEK293T cells (2 × 10⁴ cells/well), MDA-MB-231 cells, or MCF7 cells (6 × 10³ cells/well) were plated in p96. After 16–24 hours, the aptamers or the 38x(AG) unstructured ssDNA were transfected at concentrations indicated in the figure legends using Lipofectamine 2000 (Invitrogen) following to manufacturer's instructions for siRNA transfection. After 72 hours, medium was removed and 100 μl of MTT (1 mg/ml in culture medium) was added to each well, and plates were incubated at 37 °C for 4 hour. Next, 100 µl/well of lysis buffer (10% sodium dodecyl sulphate and 10 mmol/l HCl) were added and, after 24 hours of incubation, absorbance was read at 540 nm on a SpectraFluor microplate reader (TECAN). Percent inhibition was calculated relative to the cells transfected in the absence of aptamers (control).
Scratch wound-healing assays. MDA-MB-231 cells were plated in six-well plates at 5 × 10⁵ cells/well in 2 ml growth medium. After 24 hours, cells were transfected with the aptamers or the 38x(AG) unstructured ssDNA at 5 nmol/l concentration as indicated above and allowed to form a confluent monolayer for 24–48 hours. Cells were treated with 0.5 µmol/l mitomycin C for 2 hours to ensure that wounds are filled due to cell migration and not by cell proliferation.^{43 (link)} Subsequently, the monolayer was scratched with a P-10 pipette tip, washed with media to remove floating cells, and photographed with an Olympus IX70 (time 0). Plates were then incubated at 37 °C, and images were taken after 24 hours. Cells that have migrated into the wounded area were counted, and the percent inhibition of cell migration calculated relative to the control.
Colony-forming assays. For colony formation assays, MDA-MB-231 cells were plated (3 × 10⁴ cells/well) in 24-well plates. After 16–24 hours, the different aptamers or the 38x(AG) unstructured ssDNA were transfected at 20 nmol/l as above. After 16–24 hours, alive cells were counted by Trypan blue exclusion assay (Sigma-Aldrich) using the counter TC10 (Bio-Rad) and seed at 1 × 10³ cells/well in six-well plates. Approximately 8–9 days later, the colonies were fixed, stained for 30 minutes with Giemsa 0.02% (Sigma-Aldrich), and counted with a eCount Colony Counter Pen (Heathrow Scientific, Vernon Hills, IL) and a magnifying glass (×1.75) (Bel-Art Scienceware, Wayne, NJ). Percent inhibition was calculated relative to the control.
Statistical analysis. Data are presented as an average value ± SEM from three to six independent measurements in separate experiments and analyzed using Graphpad Prism 6 (San Diego, CA). The statistical significance was performed by analysis of variance followed by Tukey's test or one-sample t-test against a control value. Significance was assumed at P < 0.05.
SUPPLEMENTARY MATERIALFigure S1. Evolution of selection and relative abundance of each aptamer family.
Figure S2. Characterization of MDA-MB-231 stable lines.
Figure S3. Effects of aptamers on MNK1b kinase activity in vitro.
Figure S4. Efect of aptamers on HEK293T and MCF7 cell viability.
Figure S5. Viability and colony formation activity of MDA-MB-231 cells treated with the MNK1 inhibitors.
Table S1. Sequence and size of the selected aptamers.