NOTCH3 is a target of miR-150. (A) Sequence alignment between miR-150 and the 3′-UTR of NOTCH3 in human and mouse, as predicted by miRanda algorithm.³⁰  Solid line represents seed match region; and dashed line, deleted region. (B) Relative expression of miR-150 (left) and NOTCH3 mRNA (right) in DP vs SP CD4⁺ thymocytes, based on microarray data. The histogram reports the relative fold change (FC) in miR-150 and NOTCH3 in SP CD4⁺ compared with DP thymocytes. (C) RT-PCR for NOTCH3 in human thymocyte subsets DP, SP CD4⁺, and SP CD8⁺ (P < .05; t test). The level of expression was normalized to that of the housekeeping gene β₂-microglobulin. Shown are the average values ± SD of 2 independent experiments. (D) Western blot to detect NOTCH3 protein in human DP, SP CD4⁺, and SP CD8⁺ thymocytes. β-actin was used as a loading control. (E) Luciferase assay using LV-pre-miR-150/LV-control vectors. 293T cells were transduced with a lentiviral vector expressing pre-miR-150 (LV-pre-miR-150) or with a control vector (LV-control). After 48 hours, cells were cotransfected with a β-galactosidase expression plasmid and a luciferase reporter plasmid containing the human NOTCH3 3′-UTR (pMIR-NOTCH3) or with the parental pMIR-REPORT plasmid (pMIR-empty). (F) Luciferase assay using pCDNA_miR-150/pCDNA_empty. The 293T cells were cotransfected with the pCDNA_miR-150 expression plasmid, a β-galactosidase expression plasmid, and a luciferase reporter plasmid containing the human NOTCH3 3′-UTR (pMIR-NOTCH3), no 3′-UTR (pMIR-empty), or the miR-150-binding site-deleted human NOTCH3 3′-UTR (pMIR-NOTCH3DEL). PCDNA3.1 plasmid lacking the miR-150 fragment (pCDNA_empty) was used as a negative control. Luciferase activities were measured 30 hours after transfection and normalized to the corresponding β-galactosidase activity. Each experiment was repeated at least 3 times. *P < .05 (t test). **P < .01 (t test). ***P < .001 (t test).