Uptake and G4 Formation of ODNs

To examine the uptake of ODNs, A375 cells were placed on glass coverslips in the presence of 4 µM 5′-Cy3-labelled CpG-1-PTO and nCpG-6-PTO for 24 h. Consecutively, cells were examined using a BZ-X800 fluorescence microscope (Keyence, Neu-Isenburg, Germany). Nuclei were stained with DAPI. Formation of G4 was tested as described [22 (link),23 (link)]. Briefly, 0.2 µg 5′-Cy5-labelled ODNs were mixed with 200 and 400 ng of the G4-specific antibody BG4 (Biozol ABA-AB00174-1.1, Eching, Germany) for 15 min at room temperature. After separation by 10% non-denaturing PAGE (100 V, corresponding to 14.7 V/cm) with 0.5× TBE, fluorescence was captured using the LI-COR Odyssey gel documentation system (Bad Homburg, Germany). Likewise, specific interaction with the IFNGR was tested by incubating the Cy3-labelled ODNs with either 200 ng or 400 ng IFNGR1 (R&D Systems) or IFNGR2 (Novus Biologicals, Centennial, CO, USA) before separation by PAGE.