Human angiogenesis array

Scrambled non-silencing shRNA control or Axl knockdown MDA-MB-231 cells were seeded in a 60-mm dish. When the cells reached 50–60% confluence, the medium was replaced with serum free medium. Cell conditioned media were harvested 24 h later, centrifuged at 300 ×g for 10 min to remove cell debris. Cells from each sample were trypsinized and counted for normalization of secreted levels with total cell number. The angiogenic factors secreted in the conditioned media were measured using the human angiogenesis array (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Briefly, the blot was incubated with conditioned media. After washing and incubating the blot with HRP-conjugated secondary antibody, ChemiReagent Mix was used for chemiluminescent detection on x-ray film. The pixel density was normalized to 10⁶ cells.

Secreted VEGF and angiogenesis-related cytokines were measured in the conditioned media of MDAMB231^m cells by human VEGF Quantikine ELISA Kit (DVE00, R&D Systems) and human Angiogenesis Array (ARY007, R&D Systems) respectively.

LOX secretion was determined by Western blotting performed on concentrated CM samples using an antibody against LOX (NB100-2527, Novus Biologicals). Angiogenesis array on conditioned media was performed using the Human Angiogenesis array (R&D systems) according to the manufacturer`s protocol. For ELISA, cells derived from different tumor entities were sham-treated or irradiated and conditioned media was harvested 24 h after treatment. LOX protein levels were measured according to the protocol of the commercially available LOX-ELISA-kit (USCN Life Science Inc.). Simultaneous quantification of the number of viable cells was performed to allow correction of LOX -levels for cell number.

The relative levels of secreted cytokines into the secretome were detected using a Human Angiogenesis Array and Human XL Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA). Briefly, the secretome was incubated overnight with the membranes spotted with specific capture antibodies. Excess material was removed by washing, and the membranes were incubated with a cocktail of biotinylated detection antibodies, followed by Streptavidin-HRP. The chemiluminescent detection was performed using a Luminescent Image Analyzer LAS-3000 (FUJIFILM, Japan), and mean pixel densities of all spots were determined using TotalLab Quant software (United Kingdom). Proteins with a mean pixel density over 10,000 were considered to be highly secreted. Heatmap was generated using Morpheus software (Cambridge, MA, USA) (https://software.broadinstitute.org/morpheus).

PC9 cells transduced with lentiviruses carrying either scrambled (SCR) or ABL1/ABL2-specific (AA) shRNAs were cultured with or without MSCs for 72h followed by culture in serum-free media for another 24h. Culture supernatants were concentrated and analyzed using Human Angiogenesis Array (R&D Systems, Catalog #ARY007) following manufacturing instructions.