Figure 4.

NR4A1 regulation in A549 cells. A549 cells were treated with TGFβ alone or in combination with various inhibitors (A) or after knockdown of JNK1 pathway kinases (B), and NR4A1 mRNA levels were determined by real time PCR. (C) Identification of cis-elements on the NR4A1 gene promoter. (D) Cells were treated with TGFβ or transfected with MKK4(WT), MKK7(CA) and MKK7(DN) alone or in combination with various agents for 6 hr and then analyzed in a ChIP assay using multiple antibodies and primers targeting the −869 to −853 (F) and −669 to −86 (R) regions of the NR4A1 promoter. (E) A549 cells were treated with TGFβ (5 ng/ml) for 0, 3, 6, 9, 12 and 24 hr, and whole cell lysates were analyzed by western blots. A549 cells were transfected with oligonucleotides targeted to factors that regulate NR4A1 expression, and their effects on NR4A1 mRNA levels (F) and protein knockdown efficiencies (G) were determined by real time PCR and western blot analysis of whole cell lysates, respectively. The effects of knockdown of these same factors on cell migration (H) and intracellular location (nucleus vs. cytosol) of NR4A1 (I) was determined in Boyden chamber assays and western blots, respectively. The NR4A1 band relative to β-actin in western blot (G) was quantitated, and control values of NR4A1 were set at 1.0.