Pgl4.10 luc2 reporter vector

The two mouse ApoD genomic regions showing sequence conservation with human ApoD sequences (Fig 5) were PCR-amplified and cloned in the pCR^®II-TOPO^® vector (Invitrogen), and confirmed by DNA sequencing. These proposed promoter regions (α and β) were then digested with restriction enzymes and directionally cloned in the pGL4.10[luc2] reporter vector (Promega). Astrocyte IMA2.1 cells were Lipofectamine (Invitrogen)-transfected with the mouse ApoD promoter (α or β)-pGL4.10[luc2] plasmids. The promoter-driven expression of Luciferase was tested with the Dual-Luciferase^® Reporter Assay System (Promega) following the manufacturer’s specifications. Briefly, cells were transfected with each luciferase reporter construct and a Renilla expression vector at a 10:1 ratio. After an 18 h post-transfection period, cells were incubated for 2 h with Low Serum media (control conditions) or PQ (0.5 or 1 mM) in Low Serum media. Luminescence was then measured in cell lysates with a BMG LABTECH 96 microplate luminometer. Experiments were performed in triplicates. Promoter activities were expressed relative to Renilla activity. PQ-dependent activity was normalized to values obtained in control conditions.