Figure 1. HR12 treatment induces adherens junction organization in ras-transformed Rat1 cells. Rat1/ras cells grown in medium containing 10% FCS were exposed to 20 µM HR12, fixed, and stained with anti–ß-catenin antibody and Cy3-conjugated anti–mouse antibody. (a) No treatment; (b–d) time course of exposure to HR12: (b) 24 h, (c) 48 h, and (d) 72 h. (e) Cells were exposed to HR12 for 48 h, washed, and incubated in medium containing 10% FCS 24-h longer, before fixation. (f) Untransformed Rat1 cells are shown for comparison. Bar, 10 µm.

Figure 2. HR12-treatment induces stress-fiber formation in Rat1/ras cells. Rat1/ras cells were treated as in Fig 1 and stained with TRITC-labeled phalloidin. (a) No treatment, (b) 24 h, (c) 48 h, (d) 72 h, and (e) 48 h exposure to HR12, followed by 24 h without the inhibitor. (f) Untransformed Rat1 cells. Bar, 10 µm.

Figure 3. HR12 leads to morphological reversion of ras-transformed cells and increases dramatically cell–matrix adhesion and cell–cell contacts. Rat1 and Rat1/ras cells are compared with Rat1/ras cells that were exposed to 20 µM HR12 for 48 h. The fixed cells were stained with anti–paxillin, anti–vinculin, or antiphosphotyrosine mAb, followed by Cy3-conjugated anti–mouse antibody. Bar, 10 µm.

Figure 4. HR12 treatment of Rat1/ras cells causes elevation of adhesion molecule levels in a dose-dependent manner. Whole-cell lysates were immunoblotted with anti–ß-catenin or anti–pan-cadherin or antiphosphotyrosine (pY20) mAb. Actin levels in the Triton X-100 insoluble and soluble fractions were detected by polyclonal anti–actin antibody. The levels of ß-catenin, cadherin, phosphotyrosine, and actin were quantified using the NIH-IMAGE 1.61 program.

Figure 5. HR12 treatment of Rat1/ras cells inhibits Ras processing and affects the phosphorylation levels of MAPKs and PKB in a dose-dependent manner. Rat1/ras cells were exposed to the indicated concentrations of HR12 for 48 h, lysed, and immunoblotted with anti–Ras to allow dose–dependent inhibition of Ras processing. (up) Unprocessed Ras, (p) processed Ras. In parallel, blots were labeled with antiphosphorylated Erk (pErk), anti–Erk2 (Erk), antiphosphorylated-p38 (pp38), anti–p38 (p38), antiphospho-Jnk (pJnk), anti–Jnk (Jnk), antiphospho-PKB/Akt (pPKB), and anti–PKB/Akt (PKB). In each case, the level of phosphoenzyme was normalized to the level of total enzyme. For comparison, phosphoenzyme levels in Rat1 cells are shown (right lane).

Figure 6. Correlation between the inhibition of Ras processing and inactivation of Erk. Rat1/ras cells grown in DME containing 10% FCS were treated with 20 µM HR12 for the indicated time periods, or exposed to 20 µM HR12 for 48 h, washed, and incubated without the inhibitor 24 h longer, before lysis (wash). Lysates were immunoblotted with anti–Ras, antiphosphorylated Erk (pErk), and anti–Erk2 (Erk) antibodies.

Figure 7. Inhibition of Mek/Erk pathway by PD98059 causes morphological reversion of ras-transformed Rat1 cells. Rat1/ras grown in DME containing 10% FCS were treated for 24 h with 50 µM PD98059 or 0.05% DMSO (veh), and fixed. ß-Catenin, actin, or vinculin were labeled as described in Materials and Methods. Bar, 10 µm.

Figure 8. Activated Mek disrupts adherens junctions and stress fibers. Rat1/ras (center) and Rat1 (right) cells were transiently cotransfected with vectors carrying GFP and activated-MEK [MAPKK1({{Delta}}N3)]. 24 h after transfection, the Rat1/ras cells were treated with 20 µM HR12 for 48 h. Cells were fixed and stained with anti–ß-catenin or phalloidin. In cells that showed GFP fluorescence, indicating that they had been transfected, stress fibers and adherens junctions were disrupted. Arrowheads indicate ß-catenin–labeled adherens junctions in the nontransfected cells, while arrows indicate their loss in the MAPKK1({{Delta}}N3)-transfected cells. In Rat1/ras cells transfected with the GFP-vector alone and treated with HR12 (left), the stress fibers and adherens junctions remained intact. Bar, 10 µm.

Figure 9. Phenotypic reversion by HR12 depends on inhibition of Ras function. (a) HR12 treatment increases cell adhesion of v-ras–transformed NIH3T3 cells, but not of myristoylated-ras–transformed cells. After a 48-h treatment with 20 µM HR12 in DME + 10% FCS, the cells were fixed and stained with anti–vinculin mAb followed by Cy3-conjugated anti–mouse antibody. Bar, 10 µm. (b) Erk phosphorylation is inhibited by HR12 in NIH3T3v-ras, but not in NIH3T3myr-ras. Cell lysates were immunoblotted with antiphosphorylated-Erk mAb (pErk). The blot was stripped and reacted with polyclonal anti–Erk2 antibody. Phospho-Erk levels were normalized to total Erk levels.