In vitro T-ALL proliferation requires the activation of the NOTCH-signaling pathway. (A) Structure of the bicistronic TRIP-ΔU3-EF1α-IRES-GFP lentiviral vectors encoding or not human DL1 (left panel). Western blot analysis of DL1 expression in MS5 cells transduced with either of the 2 TRIP-ΔU3-EF1α vectors shown in the left panel. (B) Left panels: Representative growth curves observed with M18 and M30 T-ALL samples in coculture with MS5-GFP or MS5-DL1 cells. Right panels: Pictures of the leukemic cell cultures after 16 (M18) and 21 (M30) days of coculture with MS5 cells. Images were obtained using a Nikon Eclipse TE 2000-S (×200; Nikon France SAS, Champigny-Sur-Marne, France) microscope and captured using Eclipse-Net software. Images were processed using Adobe Photoshop software. (C) Amplification rate after 30 days of culture of M18 and M30. Fold amplification is referred to the initial number of plated cells. Shown are mean plus or minus SD of 6 (M18) and 3 (M30) experiments. (Inset) γ-TCR gene rearrangements of M30 at diagnosis and after 30 days of coculture with MS5 cells. (D) Phenotype of T-ALL leukemic cells before and after 30 days of culture with GFP- and DL1-expressing MS5 cells. Percentage of cells is indicated in each quadrant.