Supplementary Information
Estrogen stimulates the proliferation of human endometrial cancer cells by stabilizingnucleophosmin/B23 (NPM/B23)
Angel Chao1*§,Chiao-Yun Lin1*, Chia-Lung Tsai1, Swei Hsueh2, Ying-Yu Lin1, Cheng-Tao Lin1, Hung-Hsueh Chou1, Tzu-Hao Wang1,3, Chyong-Huey Lai1§, Hsin-Shih Wang1§
1Department of Obstetrics and Gynecology,Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, Taiwan
2 Department of Clinical Pathology,Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, Taiwan
3 Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Taiwan
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Supplementary Table 1. Characteristics of endometrial cancerpatients(for immunohistochemistry)
Number Age / Pathology / Stage152 endometrioid adenocarcinomaIIIA
254endometrioid adenocarcinomaIIIA
349endometrioid adenocarcinomaIB
461endometrioid adenocarcinomaIA
546endometrioid adenocarcinomaIA
644 endometrioid adenocarcinomaIB
762endometrioid adenocarcinomaIB
864endometrioid adenocarcinomaIB
967endometrioid adenocarcinomaI1B
1051endometrioid adenocarcinomaIA
1158endometrioid adenocarcinomaIC
1253endometrioid adenocarcinomaIB
1371endometrioid adenocarcinomaIIIC
1468endometrioid adenocarcinomaI1A
1553endometrioid adenocarcinomaIA
1660endometrioid adenocarcinomaIIIC
1753endometrioid adenocarcinomaIVA
1867endometrioid adenocarcinomaIIIC
1959endometrioid adenocarcinomaIIIA
2072endometrioid adenocarcinomaIIIC
2154endometrioid adenocarcinomaIIIC
2265endometrioid adenocarcinomaIIIC
2359endometrioid adenocarcinomaIIIC
2455 endometrioid adenocarcinomaIA
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Supplementary Table 2. Characteristics of endometrial cancerpatients(for real-time quantitative PCR analysis)
Number Age / Pathology / Stage153endometrioid adenocarcinomaIB
251endometrioid adenocarcinomaIC
353endometrioid adenocarcinomaIIIC
436endometrioid adenocarcinomaIIIA
575endometrioid adenocarcinomaIIIA
659endometrioid adenocarcinomaIB
745endometrioid adenocarcinomaII
844endometrioid adenocarcinomaIB
946endometrioid adenocarcinomaIA
1061endometrioid adenocarcinomaIA
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SupplementaryFigure 1. E2activates NPM/B23 protein expression in an ERα-dependent manner.(a) Lysates were prepared from the MCF-7 (ERα-positive) and MB-MDA231 (ERα-negative) human breast cancer cell lines.Western blotting was performed using ERαand β-actin antibodies. (b) MCF-7 and MDA-MB-231 cells were treated with E2 for 24h. Western blotting was performed using NPM/B23 and β-actin antibodies. The data shown represent themean ± standard error and were derived from three independent experiments. ＊, P < 0.05 as compared to the controls.
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(a)
(b)
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Supplementary Figure 2. Expression levels of theERαand ERβreceptors and the effects of E2, anERα-specific agonist (PPT), and anERβagonist (DPN)onendometrial cancer cells.(a)The human endometrial cancer cell lines (Ishikawa and ARK1 cells) and ERα-positive breast cancer MCF7 cellswere measured forERα expression. Equal amounts of protein lysate were separated by SDS-PAGE and subjected to immunoblotting with antibodies for ERαand β-actin. The titration concentrations of (b) Ishikawa cellstreated with E2, an ERα-specific agonist (PPT), or an ERβ agonist (DPN)areshown.(c) ARK1 cells were treated withthe ERα-specific agonist (PPT) andanERβagonist (DPN). The ARK1 cell viabilitywas less than 10% when treated with 10 μMPPT;thus, the experiment was not performed at this concentration.The data shown represent the mean ± standard error and were derived from three independent experiments. ＊, P < 0.05 as compared to the controls.
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(a) (b)
Supplementary Figure 3.
E2induces the proliferation of human cancer cells by activating NPM/B23 in more than one clone of Ishikawa and ARK1 cells.NPM/B23 knockdown in an additional(a)Ishikawa and (b)ARK1 cellclone suppressed 1 μM E2-mediated cell proliferation, asassessed by BrdU incorporation.The data shown represent themean ± standard error andwere derived from three independent experiments. ＊, P < 0.05 as compared to the controls.
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