Fig. 1

Oli-neu cultured in optimized media exhibits enhanced differentiation: (A) ICC analysis of Oli-neu cells in the various culture media stained with antibodies against β tubulin (green channel) and CNP (red channel). β tubulin allows the identification of cytoplasmic arborization while CNP localizes to the cytoplasm but is only expressed in mature OL lineage cells. Top panels – undifferentiated Oli-neu cells in proliferation medium. Middle and lower panels – Oli-neu cells cultured in standard and optimized differentiation medium, respectively. Quantification of proportion of the cells with more than two processes. Significant increases in arborization were observed in cells cultured in optimized differentiation medium. Error bars are 95% confidence intervals from more than 200 cells counted for each biological replicate, ***P < 0.001, calculated by ANOVA with post-hoc Tukey test (Scale bar − 50 µM).(B) mRNA expression of myelin specific genes, Plp1, Mbp and Cnp, and a marker for OPCs, Pdgfrα, in Oli-neu cells grown in different media. Expression of individual genes was analyzed by real time PCR and normalized to Gapdh as an internal control and plotted as a ratio of expression in undifferentiated cells. Expression was significantly increased for all three myelin specific genes but decreased for Pdgfrα in cells grown in the optimized differentiation medium. Error bars are standard error of the mean, n = 3 independent biological replicates, *p < 0.05 **p < 0.01, ***p < 0.001 analyzed by ANOVA with post-hoc Tukey test. (C). Representative immunoblot of protein lysates from Oli-neu cells cultures in the three different media. Immunoblots were simultaneously probed with anti-CNP (red) and anti-GAPDH (red) as a loading control. Quantitation of CNP relative to GAPDH show significant increases in CNP expression in cells grown in optimized medium (n = 3 independent biological replicates, **p < 0.01, ***p < 0.001 analyzed by ANOVA with post-hoc Tukey test