Figure 1. Overexpression of NMII impairs OPC branching and differentiation.

A. OPC transfected with siRNA targeting NMIIA (M2A) or NMIIB (M2B) isoforms, or non-targeting control (siCtrl) were placed in coculture with neurons and allowed to myelinate as previously described (Wang et al. 2008). After 14 days cocultures were stained with antibodies to MBP and the total number of MBP+ segments was quantified. Silencing of either isoform resulted in 1.8-2 fold increase in myelination (p<0.05; t- test). Data on graph represents the mean ± SD of two separate experiments (2 cultures/per condition/per experiment). B. Fractal analysis of OPC cultures transfected with pEGFP-N1 (pEGFP) or pEGFP-NMIIB (pM2B) and maintained in proliferating media (PDGF+bFGF) for 2 days. The mean fractal dimension (D) in control cultures was significantly higher than in OPC overexpressing NMIIB (pEFGP 1.36 ± 0.06 vs. pM2B 1.04 ± 0.04; mean ± SD; ^***p<0.0001; t-test), indicating a less complex cytoskeletal branching. Representative examples of GFP⁺-OPC with low and high fractal dimension are shown. C. Semi-quantitative analysis of OPC branching complexity. GFP⁺-OPC were classified in four categories according to the extent of branching. Overexpression of NMIIB resulted in a reduction of the percentage of OPC exhibiting medium (2) or high complexity branching (3), while increasing the percentage of cells exhibiting low complexity (1) or no branching (0). Data represent mean +/− SEM from 2 experiments (2 cultures per condition/per experiment, ^**p<0.01; ^***p<0.0001, t-test). Representative examples of low and high complexity GFP⁺- OPC are shown. D. Western blot of transfected OPC cultures kept in differentiating media (T3) for 2 and 5 days. A significant reduction (^*p<0.05, t-test) in the levels of MBP expression was found in OPC overexpressing NMIIB-GFP (asterisk) compared to control cultures (pEGFP).