Angiogenesis slides 15 well

To investigate the impact of AQP1 inhibition on vasculogenic mimicry, we treated the cells with AqB050, as well as AQP1-specific siRNA. We also used a combination of AQP1 siRNA (Cat # s1515, Life Technologies, Carlsbad, CA, USA) and bevacizumab (Avastin™, Genentech, USA) to investigate whether knockdown of AQP1 and VEGF would completely inhibit VM. Growth factor-reduced Matrigel (BD, North Ryde, NSW, Australia) was added to wells of a 15-well angiogenesis slides (Ibidi, Martinsried, Germany) and was allowed to polymerise for 30 min at 37 °C. AqB050 treatments (20 μM and 40 μM) and equivalent control DMSO (40 μM) (Sigma-Aldrich, St. Louis, MO, USA) were added to cells prior to seeding. For bevacizumab treatments, a concentration of 10 μg/mL of either bevacizumab or IgG isotype control was administered. Cell suspensions were then added to wells and were incubated for 6 h at 37 °C. Photos were taken one h and six h post incubation using AnalySIS getIT software (Olympus, Tokyo, Japan) and an F-view camera attached to the Olympus IX71 inverted fluorescence microscope (Olympus, Tokyo, Japan) with a 4× objective. Photos were saved as TIFF files and consecutive images stitched together using Photoshop CS5 software (Adobe, San Jose, CA, USA). Images were analysed for VM manually using ImageJ software (National Institute of Health, Rockville, MD, USA).