Nongenetic Reprogramming of the Ligand Specificity. of Growth Factor Receptors by Bispecific DNA Aptamers

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1 Supporting Information For Nongenetic Reprogramming of the Ligand Specificity of Growth Factor Receptors by Bispecific DNA Aptamers Ryosuke Ueki,* Saki Atsuta, Ayaka Ueki and Shinsuke Sando* Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, , Japan. Table of Contents 1. General information S2 2. Methods S Cell culture S Stimulation of A549 cells and preparation of cell lysates S Western blotting assay S ELISA assay S DU145 cell scattering assay S3 3. Sequence information S4 4. Supporting figures S5 S1

2 1. General information Reagents were purchased from standard suppliers and used without further purification. All DNA samples were purchased from Fasmac. The annealing of DNA was conducted using thermal cycler (95 C, 5 min and then cooled to 25 C at -0.1 C/s). Recombinant human HGF (#100-39) and Recombinant human PDGF (#100-14B) were purchased from PeproTech. Human alpha-thrombin (#HCT-0020) was purchased from Haematologic Technologies. The chemiluminescence images were acquired by luminescence detection system (AE-9300H, ATTO). The phase contrast images were acquired by the inverted microscope (IX-81, Olympus) equipped with the incubation chamber (INUG2F-IX3W, TOKAI HIT) and the scmos camera (Zyla-4.2-CL10-TS, Andor). 2. Methods 2-1 Cell culture A549 and DU145 cells were cultured in RPMI1640 supplemented with 10% FBS and 1% Antibiotic-Antimycotic (#15240, Life Technologies). The cells were cultured in a humidified incubator (37 C and 5% CO 2 humidified atmosphere). 2-2 Stimulation of A549 cells and preparation of cell lysates The A549 cells were seeded at 40 mm dishes and cultured in RPMI1640 supplemented with 10% FBS overnight. Subsequently, the cells were cultured in the starving medium (RPMI1640 supplemented with 0.5% BSA) for 24 h. After the starvation, the medium was replaced and then the cells were stimulated with an appropriate ligand molecule for 15 min at 37 C in the presence or absence of a bispecific aptamer. For the inhibition assay, the cells were pre-incubated with an aptamer for 15 min at 37 C, and then incubated with HGF for another 15 min at 37 C. The cells were washed with DPBS twice and lysed with lysis buffer (20 mm Tris-HCl ph 8.0, 137 mm NaCl, 2 mm EDTA, 1% Triton-X100, and 10% glycerol) supplemented with inhibitor cocktail S2

3 (# , Nacalai Tesque) and phosphatase inhibitor cocktail (# , Nacalai Tesque). The cell lysates were centrifuged at 10,000 g for 20 min at 4 C and then the supernatants were recovered. The total protein concentration of each cell lysate was adjusted to the same value according to the result obtained by microbca assay kit (#23235, Thermo Scientific Pierce). 2-3 Western blotting assay The cell lysates were separated by SDS-PAGE and then transferred to the PVDF membrane. The membrane was reacted with primary antibody at 4 C overnight. Subsequently, the membrane was reacted with secondary antibody at room temperature for 1 h. The primary antibody for phospho-met (Y1234/Y1235, #3077), Met (#8198), phospho-gab1 (Y627, #3233), phospho- ERK1/2 (T202/Y204, #4370), Phospho-Akt (S473, #4060), and Actin (#4967) were purchased from Cell Signaling Technology. The secondary antibody (Anti-Rabbit Immunoglobulins/HRP, #P0488) was purchased from Dako. All experiments were conducted three times and the similar results were obtained. 2-4 ELISA assay The assay was conducted using commercially available ELISA kit (#DYC2480, R&D systems) according to the manufacturer s instructions. The data were expressed as relative values compared with the Met phosphorylation level induced by the treatment with HGF (1 nm). The cell lysates were prepared by conducting three independent experiments and the mean values were indicated with error bars (SD). 2-5 DU145 cell scattering assay The DU145 cells were seeded at 6-well plate and cultured in RPMI1640 supplemented with 10% FBS. After 3 days, the medium was replaced with RPMI1640 supplemented with 0.5% FBS. The cells were cultured with an appropriate ligand molecule in the presence or absence of a bispecific S3

4 aptamer. After the culture plate was mounted on the microscope, the time-lapse images (30 min interval) were captured for 24 h. The migration trajectories of randomly selected cells (total 30 cells from three independent experiments) were constructed by tracking the center position of the individual cells over the observation time using Image J software ( The migration distance during the each time-lapse interval is defined as D i = Xi Xi Yi Yi 1 2, where X i and Y i represent the X position and Y position of the cell, respectively. The total migration distance (D total ) shown in Figure 3h is given as follows. D total = n i=1 D i 3. Sequence information TS1 5: GGT TGG TGT GGT TGG ATC AGG CTG GAT GGT AGC TCG GTC GGG GTG GGT GGG TTG GCA AGT CTG AT TS29: AGT CCG TGG TAG GGC AGG TTG GGG TGA CTA TCA GGC TGG ATG GTA GCT CGG TCG GGG TGG GTG GGT TGG CAA GTC TGA T PS0: CAC AGG CTA CGG CAC GTA GAG CAT CAC CAT GAT CCT GTG ATC AGG CTG GAT GGT AGC TCG GTC GGG GTG GGT GGG TTG GCA AGT CTG AT PS1: CAC AGG CTA CGG CAC GTA GAG CAT CAC CAT GAT CCT GTG TAT CAG GCT GGA TGG TAG CTC GGT CGG GGT GGG TGG GTT GGC AAG TCT GAT PS2: CAC AGG CTA CGG CAC GTA GAG CAT CAC CAT GAT CCT GTG TTA TCA GGC TGG ATG GTA GCT CGG TCG GGG TGG GTG GGT TGG CAA GTC TGA T PS3: CAC AGG CTA CGG CAC GTA GAG CAT CAC CAT GAT CCT GTG TTT ATC AGG CTG GAT GGT AGC TCG GTC GGG GTG GGT GGG TTG GCA AGT CTG AT S4

5 4. Supporting figures Figure. S1 Western blotting analysis of the phosphorylation level of Met in A549 cell lysates. The cells were pre-incubated with the aptamer ( nm) for 15 min at 37 C, and then incubated with HGF (1 nm) for another 15 min at 37 C. S5

6 Figure. S2 DU145 cell scattering assay. The cells were cultured in the presence or absence of the appropriate ligands for 24 h. The phase contrast images were captured after 24 h incubation. The parts of these images are shown in Figure 3f. Scale bars indicate 500 µm. S6

7 Figure. S3 The trajectories of cell migration observed during DU145 cell scattering assay. The cells were cultured in the presence or absence of the appropriate ligands for 24 h. The parts of these trajectories (X position = µm, Y position = µm) are shown in Figure 3g. S7

8 Figure. S4 DU145 cell scattering assay. The cells were cultured with an appropriate ligand molecule in the presence of the bispecific aptamer. The phase contrast images were captured after 24 h incubation. Scale bars indicate 500 µm. S8

9 Figure. S5 Schematic representation and western blotting analysis of (a) ssdna or (b) streptavidin-inducible Met activation. Western blotting analysis of the phosphorylation level of Met in A549 cell lysates are shown. The A549 cells were cultured in the presence or absence of appropriate ligands for 15 min. The activation of Met receptor induced by these mechanisms was also confirmed by ELISA assay (data not shown). S9