Fig. 9.

Immediate early gene 3 (ie3) cell autonomously promotes downregulation of Clr-b levels by repressing Clec2d promoter activity. a Vectors encoding the MCMV immediate early gene ORF (ie1,2,3) or the empty pIRES2-GFP vector were transiently transfected into NIH3T3 cells and then analyzed for GFP and Clr-b expression after 24 h. Dotted lines represent the secondary reagent alone; solid black lines represent Clr-b expression gated on GFP⁻ (untransfected) cells, and shaded histrograms represent Clr-b expression gated on GFP⁺ (transfected) cells. Numbers represent the fold change in Clr-b expression (GFP⁺/GFP⁻ cells). b NIH3T3 stable transfectants of Dox-inducible piggyBac vectors encoding the ie gene products or empty vector controls were induced for 2 days in 1.5 μg/mL Dox and then analyzed by flow cytometry. Dotted lines represent the secondary reagent alone; solid black lines represent Clr-b expression on untreated cells (-Dox); shaded histograms represent Clr-b expression on Dox-treated cells (+Dox). Numbers represent the fold change in Clr-b expression (+Dox/-Dox). c RNA from the cells in a was analyzed by qRT-PCR for Clec2d nascent pre-mRNA transcripts of introns 1–4 normalized relative to Tbp (n = 3 experiments). Significance was determined by a 2-tailed t test. d RNA from the cells in b was analyzed for Clec2d steady-state levels by qRT-PCR relative to Tbp (n = 3–4 experiments). e Cells as in b were transfected with luciferase-reporter vectors containing various Clec2d promoter fragments and then promoter activity was assayed by luciferase reporter assay relative to Renilla luciferase and normalized to the empty vector control (pGL3). Significance was determined by a 2-tailed t test on log-transformed values (n = 3 experiments). * p < 0.05, ** p < 0.01.