Fig. 2.

Over-expression of c-Myc stimulates MAP4K1 expression and activates c-Jun. (A) Over-expression of c-Myc increases map4k1 mRNA The mRNA levels of map4k1 and GAPDH were determined by qPCR using total RNA isolated from HEK293 cells transfected with either control or c-Myc expression plasmid for 72 h. The ratio of map4kl/GAPDH in cells transfected with the control vector is designated as 100%. Two independent experiments were performed with 3 replicates for each sample. The data are shown and expressed as mean±SD. The asterisk indicates a significant difference as determined by one-way ANOVA (P<0.01). (B) c-Myc enhances the promoter activity of MAP4K1. Increasing amounts (0–100 ng) of pCMV6-Myc plasmid and pMAP4Kl-LUC (0.2 µg) along with 10 ng of pRL-SV40 were transfected into HEK293 cells. The total DNA was maintained at 0.3 µg by adding the empty vector pcDNA 3.1 DNA The activity of cells transfected with 0 ng of pCMV6-Myc is designated as 100%. Three independent experiments were performed with 5 replicates for each sample. The represented data are shown and expressed as mean±SD (n=5). The asterisk denotes a significant difference compared to transfection with 0 µg of pCMV6-Myc as determined by one-way ANOVA (P<0.005). (C) Stimulation of map4k1 promoter by c-Myc is specific. The pcDNA pCMV-Myc, or pCMV-β-gal (0.1 µg) and pMAP4Kl-LUC (0.2 µg) along with 10 ng of pRL-SV40 were transfected into HEK293 cells as described in (B). The activity of cells transfected with pcDNA and pMAP4Kl-LUC is designated as 100%. The asterisk denotes a significant difference as determined by one-way ANOVA (P<0.005). (D) Over-expression of c-Myc elevates c-Jun phosphorylation. Cell lysate from HEK293 cells transfected with either control or c-Myc expression plasmid for 72 h was used. Western blot analysis was performed using antibodies against c-Myc, phospho-c-Jun (ser-73), and GAPDH. The ratio of phospho-c-Jun (ser-73)/GAPDH in control cells is designated as 1.0.