Figure 5. Bufexamac inhibits the tumorigenic properties of breast cancer cell in vitro.

(A)³H-thymidine incorporation was assessed in cells treated with DMSO or Bufexamac. MCF7 cells were grown under normal growth conditions with the indicated concentrations of Bufexamac for 48h. ³H-thymidine was added 4h prior to harvesting. (B) MCF-7 cell growth was assessed by trypan blue exclusion. 50,000 cells were seeded and allowed to grow for the indicated timepoints with the indicated concentrations of Bufexamac. Data are presented as mean cell number ± SEM of total live cells. (C) MCF7 cells were allowed to migrate towards 10% FBS for 24h in an 8µm pore size transwell migration assay. (D) Trypan blue exclusion assay of MCF-7 cells treated as in (B) after day 7 of treatment. (E) Immunoblot of PARP cleavage on Bufexamac treated cells. MCF-7 cells were grown in the presence of the indicated treatment for 3 days. Doxorubicin is shown as a positive control and tubulin is used as a loading control. (F) Soft agar colony formation assay of MCF-7 cells assessing a dose response to Bufexamac treatement. Colony number (G) and colony size (H) were assessed after 14 days in the presence of Bufexamac at the indicated concentrations. (I)³H-thymidine incorporation assay assessing the ability of Bufexamac to inhibit proliferation of MCF-7 cells in the presence of the constitutively deacetylated K2 HMGN2 mimic. Cells were transfected with empty vector, HMGN2, or K2R and treated with 25µM Bufexamac for 48h. ³H-thymidine was added 4h prior to harvesting and percent proliferation was calculated comparing Bufexamac treated cells to DMSO treated cells for each transfectant. All experiments were performed at least 3 times, unless otherwise noted. One-way or Two-way ANOVA with Bonferroini multiple comparisons test was used for statistical analysis where appropriate, * p<0.05; ** p<0.01; *** p<0.001.