Fig. 2
Fluorescence assays of binding of compounds to FOXM1 protein. a Direct binding of Fl-NB-72 to FOXM1 by tr-FRET (n = 2, error bars ± SD). b Representative competitive binding assays of compounds using Fl-NB-72 as the tr-FRET probe (n = 2, error bars ± SD). c DARTS assay showing increased susceptibility of FOXM1 protein to degradation by pronase upon exposure of cell extracts to NB-73. β-Actin in cell extracts is also shown for comparison. d Effect of treatment of MDA-MB-231 cells with NB-55 (5 µM), NB-73 (1.5 µM), or FDI-6 (8 µM) on FOXM1 level or e treatment of MCF7 cells with NB-55 (8 µM), NB-73 (4 µM), NB-115 (4 µM) on the cellular level of FOXM1 or FOXA1 protein (a different forkhead protein for comparison) monitored by Western blot over time. f Reversal of the downregulation of FOXM1 by cotreatment of MDA-MB-231 cells with NB-73 (1.5 or 3 µM) and the proteasome inhibitor MG132 (1 µM) for 16 h. Western blots of cell extracts are shown. Numbers shown in panels d–f are values for FOXM1 or FOXA1 corrected for β-Actin in each sample.
