Fig 2. Mechanism of action of the 3-nitropyridine compounds.

(A) 4AZA2891 and 4AZA2996 induce apoptosis in Jurkat cells. Apoptosis was measured by annexin V/PI staining. Cells were treated with different concentrations of compound or carrier (DMSO) and 24 hours later apoptotic and dead cells were stained and subsequently analyzed by flow cytometry. (B) 4AZA2891 and 4AZA2996 induce G2 cell cycle arrest in Jurkat cells. Cells were treated with different concentrations of compound or carrier (DMSO) for 24 hours and then stained with DAPI cell cycle profiling by high content imaging. Both compounds cause a clear reduction of the G0/G1 while the G2 as well as the subG1 population increases. (C) 4AZA2891 and 4AZA2996 disintegrate the microtubule network. Immunofluorescence staining of alpha-tubulin in A549 cells treated for 4 hours with 250 nM of compound 4AZA2891, 4AZA2996 or reference compound vincristine. Green: α-tubulin, blue: DAPI. Scale bar: 10 μm. (D) 4AZA2891 dose-dependently inhibits tubulin polymerization in vitro. The polymerization of purified tubulin is measured by the increase in fluorescence due to the incorporation of a fluorescent reporter into microtubules as polymerization proceeds. 4AZA2891 dose-dependently causes, as vinblastine, a marked decrease in final polymer formation as compared to the DMSO control and in contrast to the effect observed for tubulin stabilizing reference compound paclitaxel.