Sk mel 28

ABCB5⁺ MSCs (1 × 10⁵ and 1.5 × 10⁵) were seeded in two wells of a 24-well plate coated with Geltrex® basement membrane matrix (Thermo Fisher) and incubated in stem cell medium for 19–22 h at 37 °C, 3.1% CO₂. Tube structures were photographed using an inverted microscope (40× final magnification; DM IL LED, Leica, Wetzlar, Germany) equipped with a digital camera (DFC320, Leica) and semi-quantitatively classified into six (A–F) categories, ranging from A = tubular branches of several cells forming a defined network-like structure to F = no tubular branches visible (Fig. S2 (see Additional file 2)), with A–C being considered as successful angiogenic differentiation. For assay validation, human umbilical vein endothelial cells (Thermo Fisher) and human skin melanoma cells (SK-MEL-28, ATCC® HTB-72™, LGC Standards, Wesel, Germany) were used as positive and negative controls, respectively.

A375, A2058 and SK-Mel-28 cell lines were purchased from LGC Standards GmbH (Wesel, Germany). A375R, UACC-62 and Colo-800 were a generous gift from Anna Obenauf (Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria). SBcl2 was a generous gift from Prof. Beate Rinner (Department for Biomedical Research, Medical University of Graz, Graz, Austria). A2058, A375 and SK-Mel-28 cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Scientific, Vienna, Austria) supplemented with 10% fetal calf serum (FCS) (Thermo Scientific, Vienna, Austria) and 1% (v/v) penicillin/streptomycin (P/S). UACC-62 and Colo-800 cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂ in Roswell Park Memorial Institute (RPMI) 1640 (Thermo Scientific, Vienna, Austria) supplemented with 10% FCS (Thermo Scientific, Vienna, Austria) and 1% (v/v) P/S. SBCl2 cells were maintained in a similar way except that 2% FCS instead of 10% was used. For all experimental assays, FCS was reduced from 10% to 2% and from 2% to 0.5%, respectively.

Branched low molecular weight (4–10 kDa) polyethylenimine PEI F25-LMW was prepared as described previously [18 (link)]. Cell Culture media, Dulbecco’s phosphate buffered saline (PBS) and trypsin were from Sigma Aldrich (Taufkirchen, Germany), and fetal calf serum (FCS) was purchased from Gibco Thermo Fisher (Darmstadt, Germany). Cell culture plastic and other disposable plastics were from Sarstedt (Nümbrecht, Germany). Chemically synthesized antimiRs (miRCURY LNA inhibitor; Exiqon/Qiagen, Hilden, Germany or 2′-OMe modified RNAs; Eurogentec, Seraing, Belgium) were used; see Additional file 3: Table S1 for additional information. Stable human prostate carcinoma cell lines PC3, DU145 and LNCaP as well as the human or mouse melanoma cell lines LOX, A375, SK-Mel-28, B16F10 and B16V were obtained from ATCC/LGC Promochem (Wesel, Germany) or the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany).