AllStars Neg. Control siRNA (1027281) was from Qiagen and On-target plus human Notch1 siRNA smartpool was from Dharmacon (Thermo Scientific, cat# L-007771-00-0005). A total of 1.5 × 106 tumor cells (tdtomato MDA-MB-231 SORE6>GFP) were transfected with 10 µl of 20 µM siRNA stock solution using Nucleofector Kit V from Lonza (cat# VCA-1003) for 48 h, as described previously19 (link). Notch1 knockdown was confirmed using western blotting with the following antibodies: rabbit anti-Notch1 at 1 : 1000 (Cell Signaling, cat# 3608), rabbit anti-Notch2 at 1 : 1000 (Cell Signaling, cat# 5732), and mouse anti-GAPDH at 1 : 10,000 (Abcam, cat# ab8245). At 36 h post siRNA transfection, tumor cells were plated with macrophages (1 : 5 ratio) overnight. Next day, cells were imaged live on DeltaVision microscope as described above.
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