Fig. 6. Treatment of oligodendrocyte progenitors with TSA arrests the cells at an immature stage of differentiation.A, TSA prevents the expression of late oligodendrocyte differentiation markers. To test the effect of histone deacetylation on the synthesis of lineage-specific markers, progenitors were cultured for 5 d without mitogens in the absence (−TSA) or presence (+TSA) of TSA. Cells were then processed for immunocytochemistry using antibodies against A2B5 (red,a, b), O4 (red,c, d), GalC (green,e, f), and PLP (green, g, h). Cell nuclei were visualized using DAPI (blue fluorescence ina–h). Typically, after 5 d of mitogen withdrawal, control cells have lost immunoreactivity for the progenitor marker A2B5 (a) but express O4 (c), GalC (e), and PLP (g). Preventing histone deacetylation with TSA halters the progression from progenitors to mature oligodendrocytes. TSA-treated cells do not express GalC and PLP (f, h) but express O4 (d) and the progenitor marker A2B5 (b). Scale bar, 20 μm. B, TSA lowers the RNA levels of CGT and PLP. To determine whether the lack of GalC and PLP immunoreactivity in TSA-treated cells was attributable to an effect on the RNA levels, a semiquantitative RT-PCR was performed. Briefly, RNA was isolated from untreated progenitors cultured in bFGF (bFGF) or differentiated in mitogen-free medium for 1 d (1d) or 3 d (3d). For TSA-treated cultures, the inhibitor was kept in the medium for 1 d (1d + TSA) or 3 d (3d + TSA) after mitogen withdrawal. After conversion into cDNA, the same amount was amplified using primers specific for actin, CGT, and PLP.