Fig. 2.
TAM receptors expression are decreased in Cx3cr1-deficient microglia. Axl could be modulated by the transcription factor NRF2. (A) Primary cultures of microglia from Cx3cr1+/+ and Cx3cr1−/− mice were incubated with vehicle or SFN (15 μM, 6 h). Quantitative real-time PCR determination of messenger RNA levels of Axl, Mer and Tyro3 respectively, normalized by Actb (β-Actin) messenger RNA levels. Values are mean±SEM (n = 4). (B) To analyze the role of NFE2L2 in the transcriptional regulation of TAM receptors, we searched the Encyclopedia of DNA Elements at UCSC (ENCODE)25 of the human genome (Feb. 2009) for putative AREs. This database contains the experimental data from chromatin immunoprecipitation (ChIP) studies of several transcription factors. Although NFE2L2 is not included, we analysed 2 other ARE binding factors, MAFK and BACH1, for which information is available. We found evidence of MAFK or BACH1 binding in the Axl gene sequence. (C) HEK293T cells were co-transfected with NRF2ΔETGE-V5 expression vector, AXL-LUC reporter, Renilla control vector and empty vector, or treated with vehicle or SFN (5 μM for 16 h). Luciferase experiments were performed at least three times using three samples per group. (D) IMG cells were incubated in the presence of recombinant CX3CL1 (100 nM) for 4, 8 and 24 h and quantitative real-time PCR determination of messenger RNA for Axl was analysed and normalized by Actb (β-Actin) messenger RNA levels. Values are mean±SEM. Statistical analyses were performed with one-way ANOVA followed by Newman–Keuls multiple-comparison test: *p < 0.05, **p < 0.01, and ***p < 0.001, comparing the indicated groups.