Figure 6. Effect of Withaferin A (WA) treatment on leptin-induced signaling in breast cancer cells. A) Immunoblotting for pSTAT3 (Tyr705), total STAT3,

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Figure 6. Effect of Withaferin A (WA) treatment on leptin-induced signaling in breast cancer cells. A) Immunoblotting for pSTAT3 (Tyr705), total STAT3, and β-Actin using lysates from MCF-7 cells pretreated with vehicle (DMSO) or WA (1 µM) for two hours and then treated with leptin (100 ng/mL) for one hour in the absence or presence of WA. B) Quantitation of pSTAT3 (Tyr705) level for data shown in (A). Combined results from three independent experiments are shown as mean pSTAT3 (Tyr705) level relative to control with their 95% confidence intervals (error bars, n = 3 for all groups). C) Immunoblotting for pSTAT3 (Tyr705), total STAT3, and β-Actin using lysates from MDA-MB-231 cells treated with vehicle (DMSO), leptin, and/or WA as described in (A). D) Quantitation of pSTAT3 (Tyr705) level for data shown in (C). Combined results from three independent experiments are shown as mean pSTAT3 (Tyr705) level relative to control with their 95% confidence intervals (error bars, n = 3 for all groups). E) Quantitation of colony formation in MCF-7 and MDA-MB-231 cells treated with vehicle (DMSO), leptin (100 ng/mL), WA (20 or 40 nM), or leptin and WA combination for 10 days. Results shown are mean percentage of colony-forming efficiency (% of control) from three independent experiments with their corresponding 95% confidence intervals (error bars, n = 9 for all groups). Inset shows representative images for colony formation for MCF-7 and MDA-MB-231 cells. F) Representative images for migration of MDA-MB-231 cells treated with vehicle (DMSO) or leptin (100 ng/mL), WA (1 µM), or WA and leptin combination for 24 hours (×100 magnification, scale bar = 500 µm). G) Quantitation of cell migration data shown in (F). Results shown are mean cell migration (fold change) of three independent experiments with their corresponding 95% confidence intervals (error bars, n = 9 for all groups). Statistical significance of difference was analyzed by Bonferroni’s multiple comparison test. All P values were two-sided. From: Disease Subtype–Independent Biomarkers of Breast Cancer Chemoprevention by the Ayurvedic Medicine Phytochemical Withaferin A J Natl Cancer Inst. 2017;109(6). doi:10.1093/jnci/djw293 J Natl Cancer Inst | © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com. 1