E 410

OPM-2^eGFP multiple myeloma cells (2.5 × 10⁵) were mixed with rat-tail collagen and human mesenchymal stromal cells (0.5 × 10⁵) and the 1 nmol of the respective compounds. Collagen drops (30 μl) were placed on parafilm for 30 min to allow polymerization of the extracellular matrix at 37 °C. Then onplants were transferred to the CAM of 7-day-old chick embryos. After 5 days of in vivo growth, onplants were documented by the Olympus SZX10 stereomicroscope (Olympus) equipped with an Olympus DFPL 2-4x objective lens connected with a digital camera (Olympus E410) and flexible cold light (KL200; Olympus). Excised xenografts were transferred into 0.5 ml RIPA Buffer (Sigma Aldrich, Linz, Austria) and homogenized with an Ultra Turrax homogenizer three times for 5 s on ice. Thereafter, homogenate underwent three freezing/thawing-cycles in liquid nitrogen and 37 °C water bath. After centrifugation, supernatants were diluted in assay buffer. GFP levels were measured by Cell Biolabs’ GFP ELISA Kit (San Diego, CA, USA), using biotinylated anti-GFP antibodies, according to the manufacturer’s protocol.

The CAM-assay was performed as described elsewhere [69 (link)] with slight modifications. In brief, fertilized white leghorn chicken eggs (SPF eggs) were purchased from Charles River (Germany) and incubated at 37° C with 80% humidity (Grumbach BSS 160 MPGTFS) for three days. At day three, eggs were opened and transferred to plastic weighing boats. Ex ovo cultures were covered with a square petri dish and placed into a stationary incubator at 37°C and 80% humidity for six days. Then, collagen-onplants with PC3 cells were applied to CAMs (four equal onplants/CAM) and incubated for five days. Xenografts were analyzed under a stereomicroscope with a connected digital camera and flexible cold light (Olympus SZX10, Olympus E410). For histological analysis onplants were excised from the CAM, fixed in 4% paraformaldehyde solution and processed for paraffin sectioning. To determine the tumor area the ImageJ program (NIH,USA) was used. Successful target gene overexpression or downregulation was analyzed by qPCR as described above.

Liver and kidney were removed and dehydrated in graded serial of alcohol and embedded in paraffin wax [36] . Five micrometer thick sections were cut and stained by hematoxylin and eosin (H&E). One slide was prepared for each organ; each slide contained two sections and ten field areas were examined for histopathological changes. The examination was done using a light microscope (Olympus BX50) with a digital camera (Olympus E-410). The histopathological alterations in liver and kidney tissues were scored as follows: normal appearance (−), mild (+), moderate (++) and severe (+++) [36] .