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Published in final edited form as: Mol Cancer Res. 2011 Feb 2;9(3):364–374. doi: 10.1158/1541-7786.MCR-10-0526

a. Annexin V binding and propidium iodide (PI) uptake in cells treated with ESC8 were measured using flow cytometry. MCF-7, MDA-MB-231, COS-1, hepatocyte cells were treated with ESC8 (10 µM) for 16 hours or kept untreated and stained with annexin V and PI for further flow cytometric analysis. At a 10 µM concentration significantly higher levels of Annexin/PI staining were recorded in ESC8-treated MCF-7 and MDA-MB-231 cells (* p <0.05 for both An & An+PI positive, MDA-MB-231 & MCF7 cells with ESC8 treatment vs. control and * p values <0.001 between An+PI positive cancer cells with that of non cancer cells). The data shown are representative of three independent experiments with similar findings. b. The 10 µM dose of estradiol derivative ESC8 increased the BAX (an apoptosis promoter) to Bcl-2 (an apoptosis inhibitor) protein ratio in MDA-MB-231 cells after 16 hours of treatment. ESC8-treatment also up-regulated the cysolic level of cytochrome c. Bid expression did not change with ESC8 treatments. c. The 1 µM and 5 µM doses of ESC8 were sufficient for the induction of activated caspase-9 and -3 activities, respectively, in MDA-MB-231 cells after 16 hours of treatment, consistent with the increase in Annexin V/PI staining. β-Actin was used as a loading control. The relative fold expressions of protein levels have been indicated as required. Figures represent three separate experiments with similar results.

a. Annexin V binding and propidium iodide (PI) uptake in cells treated with ESC8 were measured using flow cytometry. MCF-7, MDA-MB-231, COS-1, hepatocyte cells were treated with ESC8 (10 µM) for 16 hours or kept untreated and stained with annexin V and PI for further flow cytometric analysis. At a 10 µM concentration significantly higher levels of Annexin/PI staining were recorded in ESC8-treated MCF-7 and MDA-MB-231 cells (* p <0.05 for both An & An+PI positive, MDA-MB-231 & MCF7 cells with ESC8 treatment vs. control and * p values <0.001 between An+PI positive cancer cells with that of non cancer cells). The data shown are representative of three independent experiments with similar findings. b. The 10 µM dose of estradiol derivative ESC8 increased the BAX (an apoptosis promoter) to Bcl-2 (an apoptosis inhibitor) protein ratio in MDA-MB-231 cells after 16 hours of treatment. ESC8-treatment also up-regulated the cysolic level of cytochrome c. Bid expression did not change with ESC8 treatments. c. The 1 µM and 5 µM doses of ESC8 were sufficient for the induction of activated caspase-9 and -3 activities, respectively, in MDA-MB-231 cells after 16 hours of treatment, consistent with the increase in Annexin V/PI staining. β-Actin was used as a loading control. The relative fold expressions of protein levels have been indicated as required. Figures represent three separate experiments with similar results.