GPER is involved in VEGF expression by hypoxia in SkBr3 breast cancer cells. (a) SkBr3 cells were treated for 12 h with vehicle (a2) or 100 μM CoCl2 (a3), as indicated. VEGF accumulation is evidenced by the green signal. (b) SkBr3 cells were transfected for 24 h with control shRNA (b2, b3) or shGPER (b4, b5) and treated with vehicle (b2, b4) or 100 μM CoCl2 (b3, b5) for 12 h, as indicated. VEGF accumulation was evidenced by the green signal. Nuclei were stained by DAPI (blue signal) (a1, b1). For descriptive purposes, each side panel shows the plot profiles obtained at the level of the yellow line of the corresponding inset using the program WCIF Image J for Windows. Note the higher values indicating zones of intense labeling. Images shown are representative of 20 random fields of three independent experiments.