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. Author manuscript; available in PMC: 2017 Aug 6.

Published in final edited form as: Lab Invest. 2017 Feb 6;97(6):725–736. doi: 10.1038/labinvest.2017.1

In vitro consequences of Notch3 KD in melanoma. In 1205Lu melanoma cells, exhibiting high endogenous Notch3 (Fig. 3A), Notch3 KD resulted in no effects on conventional 2D growth (A, left panel) or colony-forming ability in soft agar (B, left panels; Magnification, ×100; scale bar, 500 μm), but significantly inhibited VM in Matrigel™ tube formation assays (C; 200 ×, bar: 100 μm). When incorporated into the 2D niche co-culture model, Notch3 KD hindered EC-mediated MSLC induction and VM phenotypic switch in 1205Lu cells (D). Similarly, Notch3 KD in A375 cells, which demonstrated relatively low endogenous Notch3 expression (Fig. 3A), showed no effect on conventional 2D growth (A, middle panel) or colony-forming efficiency (B, middle panels). In contrast, in another cell lines displaying high baseline Notch3 expression, WM852, Notch3 KD led to significant growth inhibition under conventional culture condition (A, right panel), as well as 3D anchorage-independent growth in soft agar assays (B, right panels). The effect of Notch3 KD in A375 and WM852 cells on VM in in Matrigel™ tube formation assays was assessed; however, both non-target control and Notch3 KD counterparts failed to undergo VM (data not shown). *, P < 0.05.

In vitro consequences of Notch3 KD in melanoma. In 1205Lu melanoma cells, exhibiting high endogenous Notch3 (Fig. 3A), Notch3 KD resulted in no effects on conventional 2D growth (A, left panel) or colony-forming ability in soft agar (B, left panels; Magnification, ×100; scale bar, 500 μm), but significantly inhibited VM in Matrigel™ tube formation assays (C; 200 ×, bar: 100 μm). When incorporated into the 2D niche co-culture model, Notch3 KD hindered EC-mediated MSLC induction and VM phenotypic switch in 1205Lu cells (D). Similarly, Notch3 KD in A375 cells, which demonstrated relatively low endogenous Notch3 expression (Fig. 3A), showed no effect on conventional 2D growth (A, middle panel) or colony-forming efficiency (B, middle panels). In contrast, in another cell lines displaying high baseline Notch3 expression, WM852, Notch3 KD led to significant growth inhibition under conventional culture condition (A, right panel), as well as 3D anchorage-independent growth in soft agar assays (B, right panels). The effect of Notch3 KD in A375 and WM852 cells on VM in in Matrigel™ tube formation assays was assessed; however, both non-target control and Notch3 KD counterparts failed to undergo VM (data not shown). *, P < 0.05.