Splicing reporter gene constructs confirm NICD 3′UTR splicing events of noncoding NOTCH1 mutations. (A) Overall structure of the NICD splicing reporter system. The structure on the top reveals the different mutations in NICD 3′UTR together with the cryptic donor (purple) which settles in the coding region of NICD exon 34. The novel splice acceptors (red) generated by nucleotide substitution and the physiological 141 acceptor site (orange) are followed by EGFP complementary DNA (cDNA) sequences. The wt and mutant sequences are shown at the top. Only on alternative 3′UTR splicing does EGFP get spliced in frame with NICD (third panel, spliced). Thus, a NICD::EGFP fusion protein is translated and translocated to the nucleus, leading to GFP⁺ nuclei. In the case of unspliced events (lower panel, unspliced), translation of NICD is terminated by the normal stop codon (TAA) resulting in NICD⁺ but GFP^–nuclei. NICD EGFP represents an NICD::EGFP cDNA fusion construct acting as a positive Ctrl. (B) Images of immunofluorescence signals of NICD 3′UTR splicing reporter system in HeLa wt cells. NICD⁺ nuclei from different constructs demonstrated equal transfection efficiencies. GFP⁺ signals in the nuclei demonstrated 3′UTR splicing. After merging images of NICD⁺ and GFP⁺ signals with DAPI-stained HeLa wt nuclei, different splicing efficiencies of NOTCH1 noncoding mutations were displayed. Scale bar, 20 μm. (C) Quantification of immunofluorescence signals of NICD 3′UTR splicing reporter system in HeLa wt cells by ImageJ. Relative fluorescence units were normalized to signals of positive Ctrl (NICD EGFP). n = 6; mean ± standard deviation (SD); 2-tailed unpaired t test; P values are shown. (D) WBs using lysates from HEK 293 wt overexpressing NICD 3′UTR splicing reporter constructs repeated the same splicing processes and efficiencies as immunofluorescence in HeLa wt. − represents untransfected samples. Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindole; dmut, donor mutated; WB, western blot.