Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.

An array of isoforms of the nuclear estrogen receptor alpha (ER-α) protein contribute to heterogeneous response in breast cancer (BCa); yet, a single-cell analysis tool that distinguishes the full-length ER-α66 protein from the activation function-1 deficient ER-α46 isoform has not been reported. Sp...

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Autores principales:	John J Kim, Wenchuan Liang, Chi-Chih Kang, Mark D Pegram, Amy E Herr
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9775623135fe4148b38d349814c57b13
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spelling	oai:doaj.org-article:9775623135fe4148b38d349814c57b132021-12-02T20:06:24ZSingle-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.1932-620310.1371/journal.pone.0254783https://doaj.org/article/9775623135fe4148b38d349814c57b132021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0254783https://doaj.org/toc/1932-6203An array of isoforms of the nuclear estrogen receptor alpha (ER-α) protein contribute to heterogeneous response in breast cancer (BCa); yet, a single-cell analysis tool that distinguishes the full-length ER-α66 protein from the activation function-1 deficient ER-α46 isoform has not been reported. Specific detection of protein isoforms is a gap in single-cell analysis tools, as the de facto standard immunoassay requires isoform-specific antibody probes. Consequently, to scrutinize hormone response heterogeneity among BCa tumor cells, we develop a precision tool to specifically measure ER-α66, ER- α46, and eight ER-signaling proteins with single-cell resolution in the highly hetero-clonal MCF-7 BCa cell line. With a literature-validated pan-ER immunoprobe, we distinguish ER-α66 from ER-α46 in each individual cell. We identify ER-α46 in 5.5% of hormone-sensitive (MCF-7) and 4.2% of hormone-insensitive (MDA-MB-231) BCa cell lines. To examine whether the single-cell immunoblotting can capture cellular responses to hormones, we treat cells with tamoxifen and identify different sub-populations of ER-α46: (i) ER-α46 induces phospho-AKT at Ser473, (ii) S6-ribosomal protein, an upstream ER target, activates both ER-α66 and ER-α46 in MCF-7 cells, and (iii) ER-α46 partitions MDA-MB-231 subpopulations, which are responsive to tamoxifen. Unlike other single-cell immunoassays, multiplexed single-cell immunoblotting reports-in the same cell-tamoxifen effects on ER signaling proteins and on distinct isoforms of the ER-α protein.John J KimWenchuan LiangChi-Chih KangMark D PegramAmy E HerrPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 7, p e0254783 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q John J Kim Wenchuan Liang Chi-Chih Kang Mark D Pegram Amy E Herr Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.
description	An array of isoforms of the nuclear estrogen receptor alpha (ER-α) protein contribute to heterogeneous response in breast cancer (BCa); yet, a single-cell analysis tool that distinguishes the full-length ER-α66 protein from the activation function-1 deficient ER-α46 isoform has not been reported. Specific detection of protein isoforms is a gap in single-cell analysis tools, as the de facto standard immunoassay requires isoform-specific antibody probes. Consequently, to scrutinize hormone response heterogeneity among BCa tumor cells, we develop a precision tool to specifically measure ER-α66, ER- α46, and eight ER-signaling proteins with single-cell resolution in the highly hetero-clonal MCF-7 BCa cell line. With a literature-validated pan-ER immunoprobe, we distinguish ER-α66 from ER-α46 in each individual cell. We identify ER-α46 in 5.5% of hormone-sensitive (MCF-7) and 4.2% of hormone-insensitive (MDA-MB-231) BCa cell lines. To examine whether the single-cell immunoblotting can capture cellular responses to hormones, we treat cells with tamoxifen and identify different sub-populations of ER-α46: (i) ER-α46 induces phospho-AKT at Ser473, (ii) S6-ribosomal protein, an upstream ER target, activates both ER-α66 and ER-α46 in MCF-7 cells, and (iii) ER-α46 partitions MDA-MB-231 subpopulations, which are responsive to tamoxifen. Unlike other single-cell immunoassays, multiplexed single-cell immunoblotting reports-in the same cell-tamoxifen effects on ER signaling proteins and on distinct isoforms of the ER-α protein.
format	article
author	John J Kim Wenchuan Liang Chi-Chih Kang Mark D Pegram Amy E Herr
author_facet	John J Kim Wenchuan Liang Chi-Chih Kang Mark D Pegram Amy E Herr
author_sort	John J Kim
title	Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.
title_short	Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.
title_full	Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.
title_fullStr	Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.
title_full_unstemmed	Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.
title_sort	single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/9775623135fe4148b38d349814c57b13
work_keys_str_mv	AT johnjkim singlecellimmunoblottingresolvesestrogenreceptoraisoformsinbreastcancer AT wenchuanliang singlecellimmunoblottingresolvesestrogenreceptoraisoformsinbreastcancer AT chichihkang singlecellimmunoblottingresolvesestrogenreceptoraisoformsinbreastcancer AT markdpegram singlecellimmunoblottingresolvesestrogenreceptoraisoformsinbreastcancer AT amyeherr singlecellimmunoblottingresolvesestrogenreceptoraisoformsinbreastcancer
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Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.

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