Fig 2. Karyopherin alphas are widely expression in OLP and important regulators of OL differentiation.

(A) Kpna gene expression in OLP exposed only to proliferative factors (PDGFAA, 10 ng/ml and bFGF, 20 ng/ml) for 24h. Expression was quantified from isolated total RNA with NanoString nCounter Gene Expression Assay. A panel of probes was designed for target genes and normalized to housekeeping genes Alas1, Ppia, Gapdh, Actb, and Rps11. Following assay completion, raw data was normalized and analyzed using nSolverTM software. Kpna2 is the highest expressing Impα in OLP. Kpna7 is not expressed in oligodendrocytes. (B-D) Results from primary OLP pre-treated 1h with increasing concentrations the karyopherin inhibitor importazole (IPZ) or DMSO, then differentiated with T3 (40ng/ml) in the presence of IPZ/DMSO for 9h and harvested for qPCR (B) or immunocytochemistry (C). (B) Failure of karyopherin functioning in IPZ-treated cultures results in dose-dependent inhibition of differentiation as measured by a decrease in the expression of the OL maturation markers CNP or MBP in qPCR. (C,D) While, at lower doses of IPZ, inhibition of differentiation is seen in the absence of any impact on viability, higher concentrations of IPZ eventually produce apoptosis, quantified as the percentage of DAPI cells that are cleaved-caspse 3 (CC3) positive. (E-I) Extension of differentiation out to 72h with T3 in the presence of 2μM IPZ confirms this reduction in maturation. Results are from primary OLP pre-treated 1h with 2μM IPZ or DMSO, then differentiated with T3 (40ng/ml) in the presence of IPZ/DMSO for up to 72h and harvested for qPCR (E,F) or immunocytochemistry (G-I). (E,F) Failure of karyopherin functioning in 2μM IPZ-treated cultures results in inhibition of differentiation as measured by a decrease in the expression of the OL maturation markers CNP or MBP in qPCR. (G) Representative image shows maturation markers for immature/mature OL (O4), and mature OL (MBP) in the OL lineage in IPZ-treated cultures and DMSO controls treated with T3 for 72h. Maturation was assessed by quantifying %O4/DAPI (H) and %MBP/DAPI (I) expressing cells. Data presented are mean ± S.E.M. and representative of 3 (A,B,C,E,F) or 5 (H,I) independent cultures. Statistics, (B) One-way ANOVA plus Bonferroni post-test, (D,E,F) Two-way ANOVA plus Bonferroni post-test, (H,I) Student’s t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars, (C,G) 20μm. Individual data values are in S1 Data.