Journal: International Journal of Molecular Sciences

Article Title: Loss of miR-145-5p Causes Ceruloplasmin Interference with PHD-Iron Axis and HIF-2α Stabilization in Lung Adenocarcinoma-Mediated Angiogenesis

doi: 10.3390/ijms21145081

Figure Lengend Snippet: CP contributes to tumor angiogenesis. ( A , B ) The effect of CP in cell proliferation, as determined by Water Soluble Tetrazolium Salts (WST-1) and Bromodeoxyuridine (BrdU) incorporation. ( C , D ) The influence of CP inhibition on cell migration, as determined by wound healing and transwell system. ( E ) Inhibition of CP did not affect anchorage-independent cell growth of H1563 cells. ( F ) Decreased CP reduced tube formation in either a normoxic or hypoxic condition. ( G ) Inhibition of CP reduced vascular endothelial growth factor-A (VEGF-A) production. ( H ) Knockdown CP reduced angiogenesis in a mouse model. The proliferation of H1563 and CP-knockdown H1563 were measured by WST-1 and BrdU incorporation after 72 h incubation. Cells were seeded in the top of transwell insert (8 μm), and complete culture medium was added into the bottom well as chemoattractant for 48 h. The migratory cells were stained by crystal violet. Cells were stained by PKH26, and cultured in Ultralow-attachment plates for 7 days. The conditioned media of H1563 and CP-knockdown H1563 cells were collected after 48 h incubation. Human umbilical vascular endothelial cells (HUVECs) were seeded into Matrigel-coated well containing various conditioned media (50%). The tube formation was visible by Calcein-AM dye. The level of VEGF-A was determined by Luminex Assays. H1563 and CP-knockdown H1563 cells were mixed with the high concentration of Matrigel and then subcutaneously injected into nude mice (Martrigel only n = 2, others n = 6). After 28 days, the Matrigel plugs were collected and dissected, then analyzed by immunohistochemical (IHC) staining using CD34 antibody. All results are representative of at least three independent experiments and each value is the mean ± SD of three determinations; * p < 0.05. ns, not significant.

Article Snippet: The cells were stained with PKH26 dye and then seeded in ultralow-attachment plates (Corning Life Sciences, Tewksbury, MA, USA) for tumor sphere formation.

Techniques: BrdU Incorporation Assay, Inhibition, Migration, Incubation, Staining, Cell Culture, Luminex, Concentration Assay, Injection, Immunohistochemical staining, Immunohistochemistry