Figure 6: Characterization of LDR assay hits. (a) MTS Assay was performed with increasing doses of Secalonic acid D on a matched pair of lung cells: the lung cancer line HCC4017 and the normal line HBEC30KT. Secalonic acid D did not show selectivity for cancer. (b) Cells were treated with vehicle or drug and sorted by cell-cycle phase after propidium iodide staining. TSA (200 nM) and NSC-15931 (5 μM) both caused cells to accumulate in G2/M phase as compared to vehicle or 5-azadeoxycytidine (1 μM). Values do not add to 100% because of the small fraction of sub G0/G1 cells. (c) In vitro HDAC activity assays were performed on nuclear extracts treated with 10uM hit compounds (left panel). Purified active HDAC1 activity was assayed in the presence of increasing concentrations of NSC-22206, which inhibited HDAC1 (right panel) but did not inhibit the coupled reaction (right side of panel).” (d) H358 lung cancer cells were treated with drugs for 24 h and analyzed by Western Blot for changes in global histone modifications. NSC-22206 (10 μM) and TSA (200 nM) both caused an increase in levels of acetylated histone 3 whereas NSC-159631 (0.5 μM) did not.