Figure 3. LMO3/HEN2-mediated transcriptional induction of Mash1.

(A) RT-PCR. SH-SY5Y cells were infected with empty adenovirus or with the indicated combinations of recombinant adenovirus encoding HA-LMO3 or FLAG-HEN2. At the indicated time points after infection, total RNA was analyzed for expression levels of LMO3, HEN2 and Mash1 by RT-PCR. GAPDH was used as an internal control. (B) Schematic drawing of human Mash1 promoter. Nucleotide positions were indicated relative to transcriptional initiation site (+1). The putative HES1-binding sites and E-box were depicted by filled and open boxes, respectively. This genomic fragment was subcloned into appropriate restriction sites of pGL3-Basic Vector to give pluc-hMash1. (C) siRNA-mediated knockdown of LMO3 reduces the promoter activity of Mash1. SH-SY5Y cells were co-transfected with constant amount of pluc-Mash1 (100 ng) and pRL-CMV (0.2 ng) in the presence or absence of increasing amounts of expression plasmid for siRNA against human LMO3 (100 or 400 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities were measured. (D) LMO3 transactivates Mash1 promoter. Mouse neuroblastoma Neuro2a cells (1×10⁵ cells/24-well plate) were co-transfected with constant amount of pluc-hMash1 (100 ng) and pRL-CMV (0.2 ng) together with or without expression plasmid for HA-LMO3 (150 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities were measured. (E) HEN2 inhibits Mash1 promoter activity. Luciferase activities were measured in Neuro2a cells with or without FLAG-HEN2 (100 ng). (F) LMO3 interferes with negative effect of HEN2 on Mash1 transcription in Neuro2a cells. Luciferase activities were measured in Neuro2a cells transfected with HA-LMO3 (150 ng), FLAG-HEN2 (100 ng) or both of them.