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Fig. 6. Inhibition of ROCK or MLCK impairs RhoA- but not Rac3-induced cell rounding. (A) N1E-115 cells were transfected with control vector (empty vector pcDNA3.1; ev), pcDNA3.1/Rac3 or with pcDNA3/RhoA^G14V construct. After being cultured for 48 hours in the presence of 2% serum, cells were treated with the ROCK inhibitor Y-27632 (10 µM) or MLCK inhibitor ML-7 (2 µM) for 4 hours and subsequently photographed. (B) Quantification of the results shown in (A). The round cells were counted (per 100 cells) in at least two different experiments and depicted in a bar graph. (C) In contrast to LPA/RhoA-induced rounding, Rac3-triggered rounding does not involve an increase in MLC phosphorylation (MLC-P). Western blot of N1E-115 cells expressing empty vector, Rac1 or Rac3 that were cultured in 2% serum (lanes 1, 2 and 3). As positive control (Lom et al., 1993), N1E-115 cells were starved during 4 hours and subsequently stimulated with 5 µM LPA for 15 min to induce maximal MLC phosphorylation. As a loading control, both -actin and total MLC levels are shown.

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