Figure 5

LMP1 increased the binding ability of transcription factors EGFR and STAT3 to cyclin D1 promoter in vitro . (A) STAT3 binding activities within the cyclin D1 promoter were examined by EMSA. A biotin-labeled wild-type STAT3 oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type STAT3 (lane 4), unlabeled mutant STAT3 oligonucleotides (lane 5), or noncompetitive unlabeled NF-κB oligonucleotide (NS, lane 6). Biotin-labeled mutant STAT3 oligonucleotide probe was incubated with nuclear extracts of the indicated NPC cell lines (lanes 8–9). (B) Ten micrograms of nuclear extracts were pre-incubated with biotin-labeled STAT3 oligonucleotide probe in the presence of inhibitors directed against different phosphorylation sites of STAT3 (indicated above each lane). (C) The biotin-labeled wild-type EGFR oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type EGFR (lane 4), unlabeled mutant EGFR oligonucleotides (lane 6) or noncompetitive unlabeled NFκB oligonucleotide (NS, lane 7), and then EGFR DNA binding activities were examined by EMSA. (D-E) The nuclear extracts of CNE1 and CNE1-LMP1 cells were pre-incubated with biotin-labeled EGFR oligonucleotide probe in the presence of inhibitors AG1478, directed against phosphorylation of EGFR, or DNAzyme 1 (DZ1), targeting LMP1. RD: relative density.