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. 2012 Nov 20;10(11):e1001430. doi: 10.1371/journal.pbio.1001430

Figure 6. Tcf1-deficient lymphoma cells depend on Wnt and Notch signaling for their survival.

Figure 6

Several Tcf1−/− cell lines were established from Tcf1−/− thymic lymphomas, and all cell lines show Notch1 mutations and a high ratio of Lef1 long over short isoform. (A) The TCF05 cell line (phenotyped as an ISP tumor) was cultured in the absence and presence of the indicated concentrations of γ-secretase inhibitor (DAPT). Percentage of live cells was determined after 24, 48, and 72 h using flow cytometry (AnnexinV/7AAD staining). After 24 and 48 h cell cycle analysis was determined by propidium iodide staining, and the relative expression of Hes1, Deltex, Lef1-L, and Lef1-S was determined. For the cell cycle analysis, one representative example is shown and the mean expression levels relative to Abl are shown of two independent experiments. (B) Three cell lines (Jurkat, TCF05, and TCF07) were cultured for 7 h in the presence or absence of Quercetin (25 µM), and the percentage of live cells was determined by flow cytometry (7AAD/AnnexinV staining). The mean percentage (± SEM) of live cells (7AAD−/AnnV−) is shown for the three cell lines of three independent experiments. (C) The Tcf1−/− cell line TCF07 was established from a thymic lymphoma characterized as a DN3 tumor with a mutation in exon 34 of Notch1. This cell line and Jurkat cells were transfected with a GFP construct in combination with a control construct (pcDNA3) or a dominant negative Lef1/Tcf1 (pcDNA ΔNTCF) construct (used ratio GFP∶construct, 1∶10). The percentage of viable cells was determined within the transfected (GFP+) cells after 6 h. (D) HEK 293 T cells were transfected with a LEF-1 luciferase reporter plasmid containing Tcf/Lef-responsive elements. The cells were cotransfected with S33-Bcat or pCI (3 µg) or Delta N Tcf1 together with S33 B cat. To control for transfection efficiency, all transfections included the pRLTK-renilla reporter (0.15 µg). Luciferase activities are shown as mean of three independent experiments normalized to renilla activity.