Hx531

The OPCs derived from rat E16 spinal cord were seeded onto 200 µg/mL PLO (poly-L-ornithine)-coated coverslips at a density of 1×10⁵ cells/slip. For the differentiation of OPCs, PDGF/bFGF was withdrawn from the OPC medium. After this, 1% fetal bovine serum (FBS) and variable concentrations (4 μM, 2 μM, 1 μM or 0.1 μM) of HX531 (3912, Tocris Bioscience) were added, respectively, to the medium for the first 2 days to inhibit differentiation. Then, 50 nM 9-cis-RA (R4643, Sigma) was added to the OPC culture for another 3 days to promote the differentiation of OPCs into oligodendrocytes. At the end of differentiation, cells were fixed for immunocytochemical staining. Antibodies against NG2 (MAB5384, Millipore), receptor interaction protein (RIP, 1:50, gifted by Dr X M Xu, Indiana University School of Medicine, Indianapolis, IN, USA) and RXR-γ were used to identify respective cell types in the OPC culture. The OPC culture was divided into seven groups (namely Control, DMSO (dimethyl sulfoxide), 50 nM 9cRA, 0.1 μM HX531+50 nM 9cRA, 1 μM HX531+50 nM 9cRA, 2 μM HX531+50 nM 9cRA, and 4 μM HX531+50 nM 9cRA groups). Three wells were used for each group, and for each well five randomly selected fields at ×400 magnification were used for enumeration of double positive cells.