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. 2020 Sep 14;11:4607. doi: 10.1038/s41467-020-18442-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Real-time quantitative polymerase chain reaction (qRT-PCR) analysis was performed to detect the expression of IGF-1R mRNA in HCC827 cells infected with lentiviruses expressing control shRNA (sh) or the shRNA for indicated molecules, with or without osimertinib treatment, for 24 h. b qRT-PCR of IGF-1R transcripts performed in HCC827 cells, similarly treated with osimertinib as in (a), introduced with three different shRNAs for FOXA1. c HCC827 cells with control or FOXA1 shRNAs were similarly treated with osimertinib, and the indicated proteins were detected by western blotting. d The expression of IGF-1R was detected by qRT-PCR in HCC827 cells infected with the control or the FOXA1 expressing retrovirus, following similar osimertinib treatment. e The indicated proteins were detected by western blotting in the indicated cells as in (d). f HCC827 cells with FOXA1 knockdown or overexpression were cultured for 18 days in the presence of osimertinib in a 60-mm dish. The dishes were stained with crystal violet, followed by imaging. The average number of drug-resistant colonies are presented in the right panel. g HCC827 cells were treated with osimertinib (Osim) and/or cycloheximide (CHX) (50 μg/mL) for 24 h. mRNA was harvested, and FOXA1 mRNA expression was evaluated by qRT-PCR. h qRT-PCR analysis was performed to detect the FOXA1 mRNA expression in HCC827 cells, as well as IGF-1R knockdown. i The promoter region of the IGF-1R gene is shown. The blue and purple bars represent the DNase I hypersensitivity site 1 (DHS1) and the consensus FOXA1 binding site (BS), respectively. The regions covered by primer sets used for ChIP assays are indicated as red bars, named Pro0 to Pro2. TSS: transcription start site. HCC827 cells with or without osimertinib (Osim) treatment for 24 h were cross-linked and the cell lysates were prepared. ChIP analyses of H3K4me3 and H3K27Ac on the indicated regions of the IGF-1R gene are shown below. j Schematic representation of the possible mechanism of osimertinib-induced expression of FOXA1 and IGF-1R. Me:methylation. Ac acetylation. Data shown are representative of three independent experiments. Data are presented as mean ± s.d. n.s. not significant. p values are provided (two-sided Student’s t-test).