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. 2014 Aug;34(15):2822–2832. doi: 10.1128/MCB.00206-14

FIG 1.

FIG 1

Human NOTCH2 and NOTCH3 proteolytic processing and transcriptional activation are triggered by DSL ligands. (A) Coculture experiment of U2OS NOTCH3-HA cells and OP9 parental or Dll1-expressing cells in the absence or presence of GSI. DMSO was used as a vehicle control. (Top panel) HA immunoblotting of cell lysates shows expression of both the full-length (FL) NOTCH3 precursor and mature, S1-processed, NOTCH3 TMIC. Dll1 stimulation leads to activation and diminished levels of NOTCH3 TMIC and accumulation of N3EXT in the presence of GSI. (Middle panel) Dll1 immunoblotting confirms expression of Dll1, which is absent in OP9 parental cells. (Bottom panel) Lamin A/C immunoblotting serves as a loading control (Ctrl). (B) Coculture experiment of U2OS NOTCH2-HA- and NOTCH3-HA-expressing cells with OP9 parental or Jagged1 (J1)- or Jagged2 (J2)-overexpressing cells in the absence or presence of GSI. (Top two panels) Stimulation by J1 and J2 leads to activation and diminished levels of NOTCH2 and NOTCH3 TMIC. N2EXT and N3EXT accumulate in the presence of GSI. (Middle two panels) Immunoblotting confirms expression of J1 and J2, which are absent in OP9 parental cells. (Bottom panel) β-Actin immunoblotting serves as a loading control (Ctrl). (C) Coculture experiment of U2OS NOTCH3-HA cells on top of TSt-4 parental or Dll4-overexpressing cells in the absence or presence of GSI. HA immunoblotting shows that Dll4 stimulation leads to NOTCH3 receptor proteolysis diminished TMIC expression and GSI-dependent accumulation of N3EXT (short exposure). (D) Coculture experiment of U2OS NOTCH2-HA-expressing cells with Dll1 or TSt-4 parental and TSt-4–Dll4-overexpressing cells in the absence or presence of GSI. HA immunoblotting of cell lysates shows expression of both the full-length (FL) NOTCH2 precursor and S1-processed NOTCH2 TMIC. Dll1 (left) and Dll4 (right) were able to activate NOTCH2 proteolytic processing, as accumulation of N2EXT fragments could be visualized by addition of GSI. Molecular mass marker proteins are indicated. (E and F) Dual NOTCH (CSL) luciferase reporter gene activity, corrected for Renilla luciferase expression, in either 3T3 cells expressing NOTCH3 (E) or U2OS cells expressing NOTCH2 (F). In response to coculture with ligand-expressing cells, an increased transcriptional activation was observed in comparison to coculture without ligand (OP9/TSt-4), which was completely blocked by GSI treatment. NOTCH-dependent signals were in parental cells arbitrarily set to 1. Measurements correspond to at least two experiments in triplicate and are displayed as relative light units (RLU). Error bars represent means ± standard deviations.