<i>HHEX</i> is a direct transcriptional target of an LMO2 protein complex.

<p>(<b>A</b>) We analyzed the occupancy of LMO2 and its binding partners at the <i>HHEX</i> enhancer, promoter, and exon 4 (nonspecific) in LOUCY cells by ChIP. Bar graph shows a summary of 4 independent experiments using antibodies against LMO2, LDB1, LYL1, GATA3, and E47 for ChIP followed by qPCR analysis relative to IgG control. (<b>B</b>) Nonsilencing shRNA or specific shRNA directed against <i>LMO2</i> was transfected into LOUCY cells followed by sorting for GFP⁺ cells. Cells were counted and analyzed by propidium iodide flow cytometry for viability. Y-axis denotes viable GFP-expressing cells. (<b>C</b>) LOUCY cells transfected with nonsilencing (NS) or <i>LMO2</i> shRNAs were harvested 24 h after transfection and whole cell lysate prepared. Protein was separated by SDS-PAGE followed by transfer to nitrocellulose and Western blot with monoclonal anti-LMO2. Quantification was performed by infrared-dye conjugated secondary antibodies which showed specific shRNA knocked down LMO2 protein to 51% of the level present in nonsilencing shRNA. (<b>D</b>) We harvested mRNA from <i>LMO2</i> knockdown LOUCY cells and analyzed <i>MYCN</i> and <i>HHEX</i> expression by qRT-PCR. Bar graphs show quantity of <i>MYCN</i> or <i>HHEX</i> expressed relative to LOUCY transfected with non-silencing shRNA. The graph shows a summary of three independent knockdown experiments. The mean values were compared by Mann-Whitney U-test generating the <i>P</i> values shown.