Figure 4.

(A) Norstictic acid significantly stabilizes full-length Med25 in HeLa cell extracts. Cellular thermal shift assays were conducted by dosing HeLa cell nuclear extracts with 25 μM NA or equivalent DMSO and subjecting the samples to a range of temperatures. Western blot using a Med25 antibody shows increased band density in NA-dosed samples compared with the control samples, indicating thermal stabilization and target engagement. Quantification and CETSA at additional concentrations are shown in Figure S10. (B) Treatment of HeLa cells with 25 μM NA attenuates the formation of the Med25 ETV5 complex. (left) Representative Western blot showing a reduction in co-immunoprecipitation of ETV5 with Med25. (right) Quantitative assessment of ETV5 co-immunoprecipitation with Med25. The Western blot band density was measured using ImageJ and normalized by comparison to overall Med25 levels. Results are averages of biological triplicates. See Figure S11 for the blot images used. (C) NA shows positive synergy with the on-pathway kinase inhibitor lapatinib in MDA-MB-231 cells. IC₅₀ values of fixed dose ratios of NA and lapatinib were measured after 2 days using a cell viability assay (MTT) and plotted on an isobologram. See the Supporting Information for additional experimental details. (D) Analysis of MMP2 transcript levels by qPCR indicates that NA treatment decreases MMP2 levels to that of a knockout (KO) variant of the cell line. MMP2 transcript levels are normalized to the reference gene RPL19. Results shown are averages of technical triplicate experiments conducted in biological duplicate. (E) Western blot showing that treatment of VARI068 cells with NA attenuates the formation of the Med25 ETV5 complex observed via co-immunoprecipitation.