Figure 5

The RBP-J-binding motif was sufficient to induce apoptosis in Jurkat cells. (A) Full length and differentially truncated FHL1C (Additional file 7: Figure S3A) were inserted into pEGFPC1 in frame, and were used to transfect Jurkat cells. The cells were analyzed by Annexin V staining followed by FACS 48 h post-transfection. The percentages of apoptotic (Annexin V⁺) cells in the EGFP⁺ cell population were determined. (B) Jurkat cells were transiently transfected with plasmids as in (A). The numbers of EGFP⁺ cells were counted at different time points after transfection. (C) Jurkat cells were transiently transfected with plasmids as in (A). Cells were harvested 48 h post-transfection for RNA extraction. The mRNA expression levels of Hes1, Pten, Myc, p53, Bcl2, Bax, and Caspase3 were detected by qRT-PCR, with β-actin as a reference. (D) The core sequences with different length of the RBP-J-binding motif in FHL1C were fused to the 3′ terminus of EGFP in frame, to construct plasmids expressing EGFP with RBP-J-binding motif at the C-terminus. (E) EGFP containing RBP-J-binding motif inhibited NIC-mediated transactivation of RBP-J specific reporter construct. HeLa cells were transfected with different plasmids as indicated, and luciferase activity in the cell lysates was examined 48 h after transfection. (F) Jurkat cells were transiently transfected with plasmids as indicated. The cells were analyzed by Annexin V staining followed by FACS 48 h after the transfection. The percentages of apoptotic (Annexin V⁺) cells in the EGFP⁺ cell population were determined. (G) Jurkat cells were transiently transfected with plasmids as indicated. The numbers of GFP⁺ cells were counted at different time points after transfection. Bars = means ± S.D (n = 3), *P < 0.05, **P < 0.01.