Fig. 1: TRAF6 mediates EGF-induced anchorage-independent neoplastic cell transformation and cell migration.

a Ectopic TRAF6 promotes Ras (G12V)-induced foci formation. TRAF6, Ras (G12V) and TRAF6 together with Ras (G12V) were transfected into NIH3T3 cells as indicated, and a foci formation assay was performed as described in Materials and methods (upper panel). The histograms showed the result of three times repeats are calculated as means ± S.D. Significant differences were evaluated using a one-way ANOVA and the asterisk (*) indicates a significant difference (p < 0.05) (lower panel). The protein levels of transfected TRAF6 and Ras (G12V) were tested by western blotting with the indicated antibodies (lower panel). b Knockdown of TRAF6 attenuates EGF-induced cell transformation. Stable TRAF6-silenced HaCaT cells were generated as described in Materials and methods. Cells were seeded in 0.3% BME agar containing 20% FBS plus EGF (20 ng/ml) and maintained in a 37 °C, 5% CO₂ incubator for 12 d, and colonies were counted using a microscope and the ImageJ computer software program. Representative photos are shown, and data from multiple experiments are expressed as the means ± S.D. The asterisk (*) indicates a significant difference between cells expressing mock or sh-TRAF6 (p < 0.05, Student’s t-test). c, d The effect of TRAF6 on cell migration. Cells that migrated across the membrane were stained with crystal violet and imaged at ×100 magnification. The multiple areas (n = 5) represent the means ± S.D. of each group. The asterisk (*) indicates a significant difference between cells expressing mock or sh-TRAF6 (p < 0.05, Student’s t-test), as shown in c. Wound healing assays were conducted to test the migration capability in vitro as described in Materials and methods. Images (at ×40 magnification) were taken at 24 h and 36 h, as shown in d