Figure 3
TNFα stimulation leads to increased expression of active GTP-bound WT-Ras, together giving rise to CXCL8 up-regulation through the MEK pathway. (A) MCF-7 cells were transfected to express RasG12V, WT-Ras or the appropriate control vector. Cell lysates were used for RBD pull-down assays, determining the levels of activated GTP-bound Ras, and in parallel for determination of total Ras or β tubulin (loading control). The figure shows the levels of GTP-bound Ras in WT-Ras-transfected cells, not-stimulated or stimulated by TNFα (50 ng/ml; 7 min or 6 hr) or EGF (100 ng/ml; 3-4 min). The figure also shows that Ras was not detected in cells transfected with the empty control vector. The fast-migrating band of GTP-bound Ras has been detected by others [49–53], and may represent a post-translationally modified form of the protein. This band was highly expressed in the RasG12V-expressing tumor cells, and also could be minimally detected in WT-Ras-expressing tumor cells, albeit only following longer exposure (Additional file 3A). (B, C) MCF-7 cells that were transfected to express WT-Ras were not-stimulated or stimulated by TNFα (50 ng/ml) in the absence or in the presence of the MEK inhibitor PD98059 (50 μM). (B) CXCL8 mRNA levels were determined by qRT-PCR. (C) CXCL8 expression levels were determined by dual luciferase assay, using the luciferase gene under the control of WT CXCL8 promoter. Non-stimulated cells were given the value of 1. In panels A-B a representative experiment of n≥3 is presented. Panel C presents the average ± SD of n=3. *p<0.05, ***p<0.001 compared to non-stimulated cells. In Panel A, the EGF results are representatives of 3 out of 4 stimulations performed. Please see "Methods" for additional details on the experimental procedures and statistical analyses performed in this part of the study.