Fig. 1.

ZN444B is synthesized and identified as a novel coumarin-derived HDACI with potential anti-breast cancer activity. A, B The design proposal of novel coumarins containing a hydroxamate-HDACIs moiety and the chemical structure of the new HDACI, ZN444B. C, D HeLa cell nuclear extracts and MDA-MB231 cell lysates were treated with different dosages of ZN444B or SAHA, followed by co-incubation with HDAC fluorometric substrate, and HDAC activity was measured. SAHA was used as a positive control. E The enzyme inhibitory activity of ZN444B against HDAC1, HDAC2, HDAC4, HDAC5, HDAC6, HDAC8 and HDAC11. F Acetylated histone H3 (Ac-H3) and histone H4 (Ac-H4) protein level was induced by treated with indicated doses of ZN444B for 24 h in MDA-MB231 and 4T1 cell lines. Actin was used as loading control. G The statistical result of (F). All data are shown as mean ± SD, two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. H 4T1 cells were treated with ZN444B or SAHA (2 µM) for 24 h, fixed, and stained with anti-Ac-H3 or Ac-H4 (green), and DAPI (red) and visualized by microscopy (scale bar 50 μm). I  MDA-MB231 cells were treated SAHA or ZN444B for 24 h and then whole cell lysates were collected. Western blot assays were performed using indicated antibodies. J The statistical result of (I). K 4T1 and MDA-MB231 cells were treated with ZN444B in the indicated concentration, after 48 h, the MTS assay was performed. The bars indicate the mean ± SD. L 4T1 and MDA-MB231 cells were seeded on 6 well, after 12 h, cells were treated with indicated concentrations of ZN444B. On day 10, number of colonies was counted in experiments repeated three times. Results represent the average of three replications. M 4T1 cells were treated with different dosages of ZN444B for 48 h. Cell death was assessed by Annexin V/PI staining and flow cytometry. N 4T1 and MDA-MB231 cells were treated with indicated concentrations of ZN444B, and the expression of PCNA, cleaved-PARP, Bcl-2 and Bcl-XL were detected by Western blot assay with indicated antibodies. O The statistical result of (N). All data are shown as mean ± SD, two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001