Figure 2.
Figure 2. Knockdown of myosin II by shRNA results in abnormal SC morphology and prevents proper interactions with axons and myelination. (A) SC infected with lentivirus expressing shRNA against the regulatory chain of myosin II (shMLC) or a scrambled nontargeting sequence (shSCR) were seeded onto purified DRG neurons and allowed to myelinate. After 18 d, cocultures were stained for MBP and neurofilament. A clear reduction in the amount of myelin formed in cocultures by SC expressing shMLC compared with those expressing nontargeting (SCR) shRNA was consistently observed. (B, left) Validation of protein knockdown in SC cultures. Western blots from control noninfected SC cultures (NI) and cultures infected with either vector alone (V) or vectors expressing targeting (MLC) and nontargeting (SCR) shRNA showing a marked reduction in the levels of phosphorylated MLC, total MLC, and both isoforms of myosin II only in SC cultures infected with lentivirus shMLC. (right) Western blots of myelinating cocultures showing a marked reduction in the total amount of myelin proteins (P0 and MBP) in cocultures established with SC infected with shMLC. Control for vector expression (GFP) and protein loading (actin) are also shown. (C) Quantitation of MBP+ segment number and length formed in 18-d-old myelinating cocultures by SC infected with shMLC compared with nontargeting control (shSCR). Data represent mean ± SEM from two independent experiments (three cultures per condition per experiment). (D) 5-d-old cocultures stained for GFP to visualize infected SCs and also for neurofilament and laminin α2. SC expressing MLC shRNA have round nuclei and extend multiple long and thin processes. Knockdown cultures also showed a marked reduction in the expression of laminin α2 chain and impaired basal lamina assembly. In control cultures (shSCR), SC exhibited the characteristic elongated morphology upon contact with the axons. (E) Detail of an SC expressing shMLC-GFP in coculture with axons (blue). The cell has multiple processes, some of which appear to contact axons (asterisks), whereas others fail to do so (arrowheads). Bars: (A) 100 μm; (D) 100 μm; (E) 20 μm.

Knockdown of myosin II by shRNA results in abnormal SC morphology and prevents proper interactions with axons and myelination. (A) SC infected with lentivirus expressing shRNA against the regulatory chain of myosin II (shMLC) or a scrambled nontargeting sequence (shSCR) were seeded onto purified DRG neurons and allowed to myelinate. After 18 d, cocultures were stained for MBP and neurofilament. A clear reduction in the amount of myelin formed in cocultures by SC expressing shMLC compared with those expressing nontargeting (SCR) shRNA was consistently observed. (B, left) Validation of protein knockdown in SC cultures. Western blots from control noninfected SC cultures (NI) and cultures infected with either vector alone (V) or vectors expressing targeting (MLC) and nontargeting (SCR) shRNA showing a marked reduction in the levels of phosphorylated MLC, total MLC, and both isoforms of myosin II only in SC cultures infected with lentivirus shMLC. (right) Western blots of myelinating cocultures showing a marked reduction in the total amount of myelin proteins (P0 and MBP) in cocultures established with SC infected with shMLC. Control for vector expression (GFP) and protein loading (actin) are also shown. (C) Quantitation of MBP+ segment number and length formed in 18-d-old myelinating cocultures by SC infected with shMLC compared with nontargeting control (shSCR). Data represent mean ± SEM from two independent experiments (three cultures per condition per experiment). (D) 5-d-old cocultures stained for GFP to visualize infected SCs and also for neurofilament and laminin α2. SC expressing MLC shRNA have round nuclei and extend multiple long and thin processes. Knockdown cultures also showed a marked reduction in the expression of laminin α2 chain and impaired basal lamina assembly. In control cultures (shSCR), SC exhibited the characteristic elongated morphology upon contact with the axons. (E) Detail of an SC expressing shMLC-GFP in coculture with axons (blue). The cell has multiple processes, some of which appear to contact axons (asterisks), whereas others fail to do so (arrowheads). Bars: (A) 100 μm; (D) 100 μm; (E) 20 μm.

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