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. 2004 May;24(10):4487–4501. doi: 10.1128/MCB.24.10.4487-4501.2004

FIG. 3.

FIG. 3.

Dvl-dependent neurite retraction in PC12 cells. (A) Tetracycline-regulated Dvl-1 expression. PC12/Dvl cells were treated with various concentrations of tetracycline hydrochloride for 48 h, and the lysates were probed with anti-Myc and anti-GSK-3β antibodies. (B) Activation of Rho by Dvl-1 in PC12 cells. After PC12/Dvl cells were cultured in the presence (Tet +) (lanes 1, 3, 5, and 7) or absence (Tet −) (lanes 2, 4, 6, and 8) of 500 ng of tetracycline hydrochloride/ml for 48 h, the lysates were incubated with GST (lanes 5 and 6) or GST-RBD (lanes 7 and 8) and precipitated with glutathione-Sepharose. The bound proteins were probed with the anti-RhoA antibody. Aliquots of the lysates were probed withanti-RhoA and anti-Myc antibodies to show the expression levels of RhoA and Myc-Dvl-1 (lanes 1 to 4). (C) Effects of Dvl on cortical actin formation in PC12 cells. After PC12/Dvl cells were incubated with or without 500 ng of tetracycline hydrochloride/ml for 48 h, F-actin was stained with fluorescein isothiocyanate-labeled phalloidin. The arrows indicate the thickness of the ringlike structure of cortical actin filaments. (D) When the thickness of cortical actin filaments was >1 μm, the cells were counted as thick-ringlike-structure-bearing cells. The results shown are percentages of thick-ringlike-structure-bearing cells and are means ± standard errors (SE) of three independent experiments. (E) Tetracycline-regulated neurite retraction. PC12/Dvl cells were cultured in the presence [Tet (+)] (a and c) or absence [Tet (-)] (b and d) of tetracycline hydrochloride for 48 h and were further incubated with (c and d) or without (a and b) 100 ng of NGF/ml for 48 h. (F) Frequency of neurite formation in PC12/Dvl cells. After treatments with various doses of tetracycline hydrochloride and NGF, the numbers of neurite-bearing cells (as shown in panel E) were quantified. The results shown are means ± SE of three independent experiments. (G) Reversibility of Dvl-1-dependent neurite retraction by Rho-kinase inhibitor. PC12/Dvl cells cultured in the presence (a to d) or absence (e and f) of tetracycline hydrochloride for 48 h were further incubated in the presence (c to f) or absence (a and b) of NGF. Some cells (b, d, and f) were treated with 10 μM Y-27632. (H) The numbers of neurite-bearing cells shown in panel G were quantified. The results shown are means ± SE of three independent experiments.