Augmented expression of Notch and the targets in C3G-F⁺ leukemic thymocytes. (A) RNAs were extracted from sorted GFP⁺ cells from the thymi of Vect/B6, C3G-F/B6, Vect/SPA-1^−/−, and C3G-F/SPA-1^−/− HPC recipients 12 weeks after transplantation; transcripts for indicated genes were assessed by real-time PCR. The transcripts of each gene were normalized to cyclophilin; relative amounts of transcripts (folds) to those of Vect/B6 thymocytes are indicated. The means and SE of 3 to 4 mice are shown. (B) Cells from the thymi of Vect/B6 and C3G-F/SPA-1^−/− HPC recipients were lysed and immunoblotted with indicated antibodies. Full indicates full-length Notch; TM, transmembrane; and IC, intracellular Notch isoforms. Note different molecular sizes of NICD1 in Vect/B6 and C3G-F/SPA-1^−/− HPC recipients (open and solid arrowheads). (C) RNAs were extracted from sorted thymocyte subsets (DN1, DN2, DN3, DN4, and DP) from normal mice as well as total thymocytes of Vect/SPA-1^−/− and C3G-F/SPA-1^−/− HPC recipients. Notch1 (open columns) and Notch 3 (closed columns) transcripts were assessed by real-time PCR and normalized to cyclophilin; relative amounts of transcripts (folds) to those of normal DN1 cells are indicated. The means of 2 independent analyses are shown. (D) Recipients of Vect/SPA-1^−/− HPCs (open columns) and C3G-F/SPA-1^−/− HPCs (closed columns) were killed 3, 5, and 7 weeks after transplantation; GFP⁺ DN and GFP⁺ DP thymocytes were sorted using FACSVantage. RNAs were extracted and the transcripts of indicated genes were assessed by real-time PCR. Relative amounts of transcripts (folds) to those of control DN cells at 5 weeks are indicated. The means of 2 analyses are shown. NA indicates not applicable because few DP cells were developed yet.