Figure 5.

Iloprost and Fzd 9 induce phospho-ERK5 and MEF2C activity in NSCLC and is associated with PPARγ activity. (A) Retroviruses encoding stable empty LNCX/LPCX or Fzd 9 were used to transduce the H157 NSCLC cell line as described previously in Materials and Methods. Extracts were prepared from pooled G418 and puromycin-resistant cultures with MAPK lysis buffer, and aliquots containing 100 µg of protein were resolved on 10% polyacrylamide SDS gels, transferred to nitrocellulose, and probed with an antibody to phospho-ERK5 (115 kDa; Cell Signaling). The filters were stripped and reimmunoblotted for total ERK5 (115 kDa; Cell Signaling), which was used as a loading control protein. (B) The H157 cell line was transiently transfected with the MEF2C reporter, along with CMV-β-gal to normalize for transfection efficiency. MKK5 alpha DD was used as a positive control plasmid. After an overnight incubation, cells were exposed for 48 hours with 10 µM iloprost. (C) Retroviruses encoding empty LNCX were used to transduce the A549 NSCLC cell line as described previously in Materials and Methods. Extracts were prepared from pooled G418-resistant cultures with MAPK lysis buffer and aliquots containing 100 µg of protein were resolved on 10% polyacrylamide SDS gels, transferred to nitrocellulose, and probed with an antibody to phospho-ERK5 (115 kDa; Cell Signaling). The filters were stripped and reimmunoblotted for total ERK5 (115 kDa; Cell Signaling), which was used as a loading control protein. (D) The A549 cell line was transiently transfected with the MEF2C reporter, along with CMV-β-gal to normalize for transfection efficiency. MKK5 alpha DD was used as a positive control plasmid. After an overnight incubation, cells were exposed for 48 hours with 10 µM iloprost.