Figure 4

PADI inhibitor Cl-amidine inhibits proliferation in breast cancer cell lines grown in monolayer and spheroid cultures. (a) Relative mean fold difference in proliferation for the MCF10AT progression model cell lines at increasing concentrations of Cl-amidine after 5d treatment (n = 3, * p < 0.05, ** p < 0.005, *** p < 0.0005). (b) Citrulline levels for MCF10DCIS cells treated with 200 μM Cl-amidine were measured and compared to PBS control MCF10DCIS cells. Data represent (n = 3, * p < 0.005) (c) Multicellular spheroids were treated with 200 μM Cl-amidine and the diameter was measured and recorded in microns (n = 3, * p < 0.05). (d) Phase contrast images (10X) of MCF10DCIS cells grown in monolayer (2D) or multicellular spheroids (3D) treated with either vehicle (PBS) or 200 μM of Cl-amidine (scale bar = 100 μm). (e and f) MCF10A and MCF10DCIS cells were treated with different concentrations of Cl-amidine (0 μM, 200 μM, and 400 μM) and (f) 10μg/mL Tunicamycin, and analyzed by flow-cytometry. Data represents percent apoptotic cells (cleaved Caspase-3 positive) or percentage of cells in various phases of the cell cycle (DAPI), and are expressed as the mean ± SD from three independent experiments (* p < 0.005, ** p < 0.005).