Fig. 4.

Macrophage induction of COX-2 in breast cancer cells is dependent on MAPKs and cJun. (A) HCC1954 cells were cocultured with THP-1 cells or treated with 1 h coculture CM and levels of phospho-ERK (p∼ERK) and ERK in HCC1954 cell lysates were determined by western blot. (B) HCC1954 cells were cocultured with THP-1 cells or treated with 1 h CM in the absence or presence of PD98059 for 6 h and levels of COX-2 and β-actin in HCC1954 cell lysates were determined. (C) HCC1954 cells were cocultured with THP-1 cells or treated with 1 h CM. Cell lysates were then analyzed for p38 MAPK activity by measuring the phosphorylation of activating transcription factor (p∼ATF). (D) HCC1954 cells were cocultured with THP-1 cells or treated with 1 h coculture CM in the absence or presence of SB202190 for 6 h. (E) HCC1954 cells were cocultured with THP-1 cells or treated with 1 h coculture CM. Levels of phospho-JNK (p∼JNK), phospho-cJun (p∼cJun), cJun and β-actin in HCC1954 cell lysates were determined utilizing western blot. (F) HCC1954 cells were cocultured with THP-1 cells or treated with 1 h coculture CM in the absence or presence of JNK inhibitor for 6 h and levels of COX-2 and β-actin in HCC1954 cell lysates were determined. (G) HCC1954 cells were cocultured with THP-1 cells for 1 h. Chromatin fragments were immunoprecipitated with anti-p∼cJun, and the COX-2 promoter was amplified by real-time PCR. The COX-2 promoter was not detected when normal IgG was used (data not shown). Data are mean ± standard deviation (n = 7; *P < 0.05). (H) HCC1954 cells were transfected with siRNA-targeting cJun for 48 h and then cocultured with THP-1 cells for 6 h. Levels of cJun, COX-2 and β-actin in HCC1954 cell lysates were determined by western blot. (I) HCC1954 cells were cocultured with THP-1 cells or treated with 1 h coculture CM in the absence or presence of ATRA for 6 h, and levels of COX-2 and β-actin in HCC1954 cell lysates were determined.