A. SiHa cells were transfected with vector alone or Bcl-XL–EGFP, Bcl2- EGFP and ER-Bcl2. The whole cell extract prepared from the cell was probed for Bcl2 and Bcl-XL. Beta actin is served as the loading control. (B). The above panel cell lines were treated with zerumbone 50 μM and 100 μM for 24 h. Then the cells were stained with Hoechst to quantify chromatin condensation. The results shown is average ±SD (n = 4)(**p≤001). (C). The whole cell extract prepared from vector alone or BclXL–EGFP, Bcl2 EGFP and ERBcl2, untreated, or treated with zerumbone 50 μM and 100 μM for 24 h were probed with antibodies against caspase 8, hsp70, Bid, Bax, caspase 3 by western blot technique. Beta actin served as loading control. (D). The whole cell extract prepared from vector alone or Bcl-XL–EGFP, Bcl2 EGFP and ER-Bcl2, untreated, or treated with zerumbone 50 μM and 100 μM for 24 h were probed with antibodies against hsp90, Akt, cyclin D1, XIAP, Survivin, PUMA, by western blot technique. β-actin and hsc70 served as loading control. (E). SiHa vector alone, BclXL–EGFP, Bcl 2 EGFP and ERBcl2, untreated, or treated with zerumbone 50 μM were stained with Cell ROX Red as described and analysed by flow cytometer. (F). SiHa vector alone, BclXL–EGFP, Bcl 2 EGFP and ERBcl2, untreated, or treated with zerumbone 50 μM were stained with t-BOC as described and analysed by flow cytometer. (G). MCF-10 A, Human Mammary epithelial cells, human smooth Muscle cells and endothelial progenitor cells were treated with zerumbone 50 μM for 24 h. Then the cells were stained with TMRM or DCF-DA as described and analysed by flow cytometer. (H). Endothelial progenitor cells and MCF10A cells were stained with TMRM and Hoechst followed by zerumbone50 μM treatment. The wells were repeatedly imaged at the indicated time points.

https://doi.org/10.1371/journal.pone.0273729.g001