Figure 4

Inhibitors and dominant negative mutants targeting the EGFR and STAT3 pathways attenuated LMP1-augmented cyclin D1 promoter activity. (A-B) Stable expression of EGFR-DN and STAT3β inhibited the LMP1-increased activity of cyclin D1. The indicated NPC cell lines were transfected with a cyclin D1 promoter-reporter construct, a Renilla luciferase transfection control plasmid, and an EGFR-DN or STAT3-β expression plasmid. Twenty-four hrs. after transfection, the cells were treated with DNAzymes or a control oligo (2 μM) for 12 hrs. Cells were harvested at 36 hrs. after transfection and subjected to the luciferase assay. Firefly luciferase was measured and normalized to Renilla luciferase activity. The results were expressed as fold induction of the reporter activity in vector-transfected CNE1 cells, which was assigned a value of 1. (mean ± SD, n =3, *p < 0.05) (C) WHI-P131, PD98059 and AG1478 inhibited the activity of cyclin D1 induced by stable expression of LMP1. CNE1-LMP1 cells were transfected with a cyclin D1 promoter-reporter construct and a Renilla luciferase plasmid as an internal control. Twenty-four hrs. after transfection, the cells were treated with WHI-P131, PD98059, AG1478 or 0.1% DMSO for 2 hrs. The cells were harvested at 26 hrs. after transfection and subjected to the luciferase assay. An empty firefly reporter vector served as a control (n = 3). * p < 0.05. (D) WHI-P131, PD98059 and AG1478 inhibited the expression of cyclin D1 induced by stable expression of LMP1. The cells were harvested for Western Blot at 8 hrs. after the treatment of WHI-P131, PD98059, AG1478 or 0.1% DMSO. β-actin was served as an internal control.