Fig. 2: TRAF6 affects the cSCC malignant phenotype.

a Knockdown of TRAF6 attenuates the proliferation of A431 cells. TRAF6-knockdown cells were generated by using different sequences as described in Materials and methods. TRAF6 expression was assessed by western blotting as indicated (upper panel). Histograms indicated relative TRAF6 expression, 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). b The TRAF6-silenced cells were seeded into 96-well plates, and proliferation was assessed by using a CellTiter96 Aqueous One Solution detection kit. The data from multiple experiments are expressed as the means ± S.D. Significant differences were evaluated using one-way ANOVA, and the asterisk (*) indicates a significant difference (p < 0.05) (upper panel). The effect of TRAF6 knockdown on cell cycle distribution. sh-Mock, sh-TRAF6#1&#2 cells were analyzed by FCM as described in Materials and methods (lower panel). c The protein level and transcriptional expression of p21 were measured by western-bloting and RT-real time PCR as described in Materials and methods. The data from triplicate experiments are expressed as the means ± S.D. Significant differences were evaluated with one-way ANOVA, and significant differences are indicated, as shown in (c). d, e 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 48 h, as shown in (d). The effect of TRAF6 on cSCC cell migration. Cells that migrated across the membrane were stained with crystal violet and imaged at ×100 magnification. Data represent the means (n = 3) ± 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 (e)