Figure 1
Rho/ROCK pathway activation and morphology. (a) Chondrocytes were cultured with vehicle (control) or 10 ng/ml TGFα for up to 30 min. RhoA G-LISA assay was used to assess levels of GTP-bound RhoA. Mean (N=4 independent isolations) relative light units (RLU) (plus s.d.) are shown. Significantly different means (P<0.05) compared with controls are indicated by asterisks. (b) Protein was isolated from chondrocyte cultures cultured for 15 or 30 min with vehicle only (control) or with 10 ng/ml TGFα. Levels of phosphorylated LIM kinase (phLIMK1/2) and myosin light chain (phMLC2) were assessed relative to total LIMK1/2, MLC2 and β-actin levels by Western blot. (c) Immunostaining for phLIMK1/2 and phMLC2 (brown) and hematoxylin counterstaining of tissue sections from explant cultures incubated for 5 days with or without (control) 10 ng/ml TGFα. Scale bar represents 50 μm. (d) Chondrocytes were cultured with vehicle (DMSO) or 10 ng/ml TGFα for 48 h in the presence or absence of 10 μM ROCK (Y27632), 1 μg/ml RhoA (C3) or 10 μM MEK1/2 (U0126) inhibitor. Cells were fixed and stained with phalloidin–rhodamine for actin (red signal) and Toto-3 iodide for nuclei (green signal) and imaged by confocal microscopy. Scale bar represents 20 μm.
