br Immunoblot Analysis br Total
Total cell lysates were prepared in JS buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 5 mM EGTA, 1 mM Na3VO4, protease inhibitors) and then boiled in SDS/b-mercaptoethanol sample buffer. Proteins were separated by electrophoresis and then blotted onto polyviny-lidene difluoride membranes (Millipore, Billerica, MA, USA) by an electrophoretic transfer. After blocking with 5% dried milk in Tris-buffered saline (TBS) containing 0.1% Tween 20, membranes were incubated at 4 C overnight with the following primary antibodies: cyclin-dependent kinase 6 (Cdk6; Santa Cruz Biotechnology, Stock-ton, CA, USA); phospho-retinoblastoma (pRb; Cell Signaling Tech-nology, Danvers, MA, USA); cyclin D1 (Cell Signaling Technology, Danvers, MA, USA); phospho-Axl (pAxl; Cell Signaling Technol-ogy, Danvers, MA, USA); Axl (R&D Systems, Minneapolis, MN, USA); a-tubulin (Santa Cruz Biotechnology, Stockton, CA, USA); vinculin (Cell Signaling Technology, Danvers, MA, USA). Ligand-dependent Axl receptor activation was performed by using Gas6 from R&D Systems. Cells were serum starved overnight, pre-treated with 200 nmol/L GL21.T, CtrlApt, or GL21.T-137 for 3 h, and then stimulated with 400 ng/mL Gas6 either alone or in pres-ence of each aptamer or complex. Blots were quantified with ImageJ software.
Cells were seeded in 96-well plates (3 103 cells/well) and transfected with miR-137 or ctrl-miR or treated with GL21.T, control aptamer, control complex (CtrlApt-137) or GL21.T-137 (400 nmol/L final concentration) for 72 h. For combined regimes, 1 mmol/L Paclitaxel or 1 mmol/L Erlotinib (Sigma, St. Louis, MO, USA) were used. Cell viability was assessed by CellTiter 96 proliferation assay (Promega, Madison, WI, USA) and expressed as percent of viable treated Latrunculin A with respect to control untreated cells.
Cells were seeded in 6-well plates (1.4 105 cells/well) and trans-fected with miR-137 or ctrl-miR or treated with 400 nmol/L GL21.T, control aptamer, CtrlApt-137, or GL21.T-137 as indicated. After 24 h, cells were trypsinized, resuspended in medium serum free, and counted. Cells (1 105 in 100 mL serum-free medium per well) were then plated into the upper chamber of a 24-well transwell (Corning Incorporate, Corning, NY, USA) and exposed to 10% FBS as inducers of migration (0.6 mL, lower chamber). After 24 h, migrated cells were visualized by staining with 0.1% crystal violet in 25% methanol. The percentage of migrated cells was evaluated by eluting crystal violet with 1% SDS and reading the absorbance at 594-nm wavelength. Molecular Therapy: Nucleic Acids
In Vivo Animal Study
A549 cells (5 106/mouse) were injected subcutaneously into flanks of 8-week-old NOD/SCID nude mice (Charles River). When tumor masses reached about 3 mm in length), mice were divided into three groups of three and intra-peritoneal injected with either PBS, GL21.T, or GL21.T-137 (1600 pmol in 100 mL/injection, three injections/ week). After 1 week, tumor masses were measured by microcallipers and calculated according to the following formula: tumor volume (mm3) = L W2/2, where L is the length and W is the width. Animals were then scarified and tumors were excised and stored in 10% paraffin for histological examinations. Sections of 4–5 mm of tumors were deparaffinized and stained with H&E.
The animal experiments were approved by the Animal Ethics Committee of the Italian Ministry of Health.
Statistics were analyzed with the Student’s t test for comparisons between two groups or by ANOVA for multiple analyses.
Supplemental Information can be found online at https://doi.org/10.
S.N. and S.C. designed and performed the majority of the experi-ments, interpreted results, and assisted with manuscript preparation. M.C. performed and/or assisted with several experiments. A.F. pro-vided human lung biopsies. P.S. provided useful reagents and advice. F.d.N. and S.B. performed and contributed to interpreting the in vivo experiments. C.L.E. coordinated the research, secured the funding, and guided the experimental design and the preparation of the manuscript.
CONFLICTS OF INTEREST
The authors declare no competing interests.
We wish to thank V. de Franciscis for suggestions and L. Baraldi and F. Moscato for technical assistance. This work was supported by funds from the Italian Ministry of Health (GR-2011-02352546 to C.L.E.) and TERABIO.
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