Us qualified fetal bovine serum

Human ovarian cancer cell line A2780/CP70 and human normal ovarian cells IOSE-364 were cultured in RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% US-qualified fetal bovine serum (Invitrogen, Grand Island, NY, USA) at 37 °C with 5% CO₂. HUVECs were cultured in vascular basal medium supplemented with endothelial cell growth kit-VEGF (ATCC, Manassas, VA, USA) at 37 °C with 5% CO₂.

The platinum-resistant human ovarian cancer cell line OVCAR-3 was kindly provided by Dr Jiang of West Virginia University. OVCAR-3 cells were cultured in RPMI 1640 medium (Sigma, St Louis, MO, USA) supplemented with 20% US-qualified fetal bovine serum (Invitrogen, Grand Island, NY, USA) at 37 °C with 5% CO₂. To culture spheres of cells, OVCAR-3 cells were harvested and seeded at a density of 2 × 10⁴ cells per well in 6-well ultra-low attachment plates (Corning, NY, USA) in Mammocult complete medium (STEMCELL Technologies, Vancouver, BC, Canada) for 7 days. Spheres were transferred to the next generation by dissociation into single cells with Accutase (STEMCELL Technologies, Vancouver, BC, Canada) and seeded at a density of 2 × 10⁴ cells per well. Third-generation spheroid (SP) cells were used for all the following experiments.

Human ovarian cancer cell line A2780/CP70 was kindly provided by Dr. Bing-Hua Jiang from Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV, USA. Cells were cultured in RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% US-qualified fetal bovine serum (Invitrogen, Grand Island, NY, USA) at 37 °C with 5% CO₂.

Human ovarian cancer cell lines, OVCAR-3 and A2780/CP70, were kindly provided by Dr. B. Jiang, Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV, USA. Cells were maintained in RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% US-qualified fetal bovine serum (Invitrogen, Grand Island, NY, USA). HUVEC cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in a vascular cell basal medium (ATCC) supplemented with Endothelial Cell Growth Kit-VEGF (ATCC). All cells were maintained in a humidified incubator with 5% CO2 at 37 °C. Galangin (purity: 98%) was purchased from Shanghai yuanye Bio-Technology (Shanghai, China). Myricetin (purity: 97%) was purchased from J & K Chemical Technology (Beijing, China). Galangin and myricetin were dissolved in dimethyl sulphoxide (DMSO) to make stock solutions of 100 mM. The primary antibodies against phospho-Akt (Ser473) (p-Akt), total-Akt (t-Akt), phospho-ribosomal protein S6 kinase (p-p70S6K), total-p70S6K (t-p70S6K), and p21 were purchased from Cell Signaling Technology, Inc. (Dancers, MA, USA). The primary antibodies against p53, NFκB (p50), PTEN and GAPDH were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). HIF-1α, mAkt, p70S6K plasmids and VEGF, HIF-1α reporter were purchased from Addgene (Cambridge, MA, USA).