Regulation of RAR-b and RXR-c in Schwann Cells Figure 2. RXR-c levels in SCs are induced by axonal contact mimickers. (A–D) Isolated SCs were adapted for 24 hours to the absence of the axonal contact mimickers BPE and forskolin and subsequently treated for 48 hours with 2 mM forskolin (Fk), 60 mg/ml BPE or the combination of both, and the levels of RXR-c (A), RAR-b (B), MAG (C) and Egr2 (D) were analyzed by Q-RT-PCR. In all instances, the levels reached after the acute combined treatment were compared to SCs continuously grown in the presence of BPE and Fk (BPE+Fk always). (E–F) SCs were adapted for 24 hours to the absence the axonal contact mimickers and subsequently treated for 48 hours with 2 mM forskolin (Fk), 5 mM cyclopamine (CPM) or the combination of both, and the levels of RXR-c (E) and RAR-b (F) were analyzed by Q-RT-PCR. All values are shown as the mean 6 SD relative to their respective controls (cells grown in the presence of vehicle). Significance was assessed by unpaired Student’s t-test. n.s.: non-significant; *: p,0.05; **: p,0.01; ***: p,0.005. doi:10.1371/journal.pone.0017023.g002 affinity to RAR and RXR, or with the pure RAR synthetic agonist TTNPB that only activates RAR. All three retinoids were equally able to induce RAR-b and down-regulate RXR-c mRNA levels to a similar extent, which indicates that binding to RAR is sufficient to mediate the effect of retinoic acid (Fig. 7A). All three retinoids were also able to regulate to a similar extent both genes in purified primary Schwann cell cultures (Fig. 7B), although TTNPB was more potent, specially regarding RAR-b up-regulation, indicative of its higher affinity for RAR binding compared to the natural retinoids [32]. At the low concentration of 1 nM, TTNPB already produced a statistically significant down-regulation of RXR-c and upregulation of RAR-b, while 9-cis-RA or all-trans-RA only produced a small regulation of both receptors that was not statistically significant. At the more elevated but still physiologically PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e17023