Figure 7. NR4A1 localized mainly in the nucleus during autophagic cell death, and is transcriptionaly competent.

HEK293 cells were transfected with NK₁R and exposed to SP for the indicated time. A, Immunofluorescence of NR4A1 (red) showed it mainly in nuclei, which were stained with Sytox Green. 20× magnification. B, Examples of cells where NR4A1 (red) was found by immunofluorescence in both the cytoplasm and the nucleus (DAPI) after 3 hr of exposure to SP. 40× magnification. C, Luciferase essay to quantify NR4A1 transcriptional activity driven by either NBRE or NuRE containing promotor. Folds of luciferase activity after 6 hr of SP addition with respect to control cells are plotted. Bar represent standard deviation (n = 4, ***, p<0.001). D, NR4A1 interacts with p53 but not with Beclin 1. HEK293 cells were transfected with His-tagged NR4A1 and NK₁R, and exposed or not to SP. NR4A1 was pulled down by NiNTA, and the presence of Beclin 1 or p53 complexed with NR4A1 was analized by Western blot.