Figure 1.
Figure 1. Inhibition of myelination in SC cocultures treated with blebbistatin. (A) 2-wk-old myelinating SC neuronal cocultures stained for MBP, Krox-20, and neurofilament. A significant reduction in myelin formation and Krox-20 expression was observed in cultures treated with the active (−) blebbistatin enantiomer compared with control cultures and cultures treated with the inactive (+) enantiomer. (B) Myelinated segments formed in blebbistatin (−)-treated cultures (right) were shorter than in control cultures (left) and frequently exhibited rounded and off-centered SC nuclei (asterisks). (C) Quantitation of internode length and number in 2-wk-old cocultures. A dose-dependent reduction in both the total number (n) and the length of the myelinated segments was consistently observed in treated cultures compared with the control. At the highest dose of blebbistatin (50 μM), myelination was completely abolished. Data in the graph represent mean ± SEM from two independent experiments (two cultures per condition per experiment). ***, P < 0.0001. (D) Western blots from 2-wk-old cocultures, which confirm a significant reduction in the expression of myelin proteins, transcription factors, and laminin chains in blebbistatin-treated cultures (T). Actin and Tau levels were comparable in both treated and control cultures. (E and F) Ultrastructural analysis of 18-d-old myelinating cocultures treated with 25 μM blebbistatin showing abnormalities in axon ensheathing and segregation. (E) An SC on top of a bundle of axons appears arrested at a premyelinating stage. Many axons (asterisks) are surrounded by very short cytoplasmic processes (black arrowheads), which fail to completely wrap or segregate them in a 1:1 association. Rough endoplasmic reticulum (red arrowheads) is very prominent in this cell. (F) A large segregated but nonmyelinated axon (ax), which is also contacted by a process (red asterisk) derived from a different SC. Many large-diameter naked axons are also observed in this field (black asterisks). The arrowhead points to a very short cytoplasmic process failing to surround an axon. Bars: (A) 100 μm; (B) 25 μm; (E and F) 1 μm.

Inhibition of myelination in SC cocultures treated with blebbistatin. (A) 2-wk-old myelinating SC neuronal cocultures stained for MBP, Krox-20, and neurofilament. A significant reduction in myelin formation and Krox-20 expression was observed in cultures treated with the active (−) blebbistatin enantiomer compared with control cultures and cultures treated with the inactive (+) enantiomer. (B) Myelinated segments formed in blebbistatin (−)-treated cultures (right) were shorter than in control cultures (left) and frequently exhibited rounded and off-centered SC nuclei (asterisks). (C) Quantitation of internode length and number in 2-wk-old cocultures. A dose-dependent reduction in both the total number (n) and the length of the myelinated segments was consistently observed in treated cultures compared with the control. At the highest dose of blebbistatin (50 μM), myelination was completely abolished. Data in the graph represent mean ± SEM from two independent experiments (two cultures per condition per experiment). ***, P < 0.0001. (D) Western blots from 2-wk-old cocultures, which confirm a significant reduction in the expression of myelin proteins, transcription factors, and laminin chains in blebbistatin-treated cultures (T). Actin and Tau levels were comparable in both treated and control cultures. (E and F) Ultrastructural analysis of 18-d-old myelinating cocultures treated with 25 μM blebbistatin showing abnormalities in axon ensheathing and segregation. (E) An SC on top of a bundle of axons appears arrested at a premyelinating stage. Many axons (asterisks) are surrounded by very short cytoplasmic processes (black arrowheads), which fail to completely wrap or segregate them in a 1:1 association. Rough endoplasmic reticulum (red arrowheads) is very prominent in this cell. (F) A large segregated but nonmyelinated axon (ax), which is also contacted by a process (red asterisk) derived from a different SC. Many large-diameter naked axons are also observed in this field (black asterisks). The arrowhead points to a very short cytoplasmic process failing to surround an axon. Bars: (A) 100 μm; (B) 25 μm; (E and F) 1 μm.

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