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. Author manuscript; available in PMC: 2009 Feb 1.
Published in final edited form as: Neurobiol Dis. 2007 Sep 19;29(2):278–292. doi: 10.1016/j.nbd.2007.09.002

Fig. 7. Merlin suppression by RNAi in arachnoidal cells demonstrated phenotypes similar to merlin(-) meningioma cells.

Fig. 7

Total protein lysates were harvested from confluent AC006 arachnoidal cells 72 hrs after infection of control mock (con), scr or merlin-specific (meri-5 and meri-6) RNAi viral supernatants. (A) Immunoblot analysis of merlin protein expression in control and RNAi infected arachnoidal cells demonstrated that merlin expression was reduced substantially in response to meri-5 and meri-6 RNAi infections compared to con or scr RNAi. No consistent change in ERM protein expression levels was detected upon merlin downregulation. GAPDH expression was used as a control for equal protein loading. (B) Expression and activation of Erk and cyclin D1 in response to merlin RNAi-mediated downregulation were examined by immunoblot analysis. Merlin knockdown arachnoidal cells exhibited a slight, but reproducible increase of 1.21 ± 0.05, p=0.003 (mean fold ± SD) in Erk1/2 activation compared to control scr RNAi cells. Data were derived from multiple experiments (n = 3) and values were normalized to GAPDH levels to adjust for total protein loading. Shown are representative blots from a single experiment. There was no significant difference for cyclin D1. (C) Merlin RNAi arachnoidal cells exhibited a statistically significant decrease of 2.21-fold (0.45± 0.13, p=0.002) in α-catenin expression levels relative to scr RNAi treated cells. Catenin protein levels were normalized to GAPDH to adjust for total protein loading, and values for mock control infected cells were arbitrarily set to 1.0. Data represent mean expression levels from multiple experiments (n = 4). Shown is a representative immunoblot. (D) Merlin knockdown (meri-5) in arachnoidal cells resulted in increased lamellipodia formation (arrowheads) and disorganized, short, dense stress fiber bundles that terminated at numerous focal contacts scattered throughout the cell. In contrast, scr RNAi arachnoidal cells did not exhibit prominent lamellipodial protrusions and displayed a more organized and filamentous stress fiber phenotype compared to merlin RNAi arachnoidal cells. Scr RNAi arachnoidal cells demonstrated focal contacts at the cell periphery and a marked reduction in the number and thickness of stress fiber bundles. Fixed cells were stained for F-actin and paxillin as described in Materials and Methods (Bar, 20 μm).