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. 2020 Jan 17;11:338. doi: 10.1038/s41467-019-14219-6

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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. 1 — a LNCaP (blue circles), C4-2B (green triangles) and LNCaP-AI (red squares) cells were cultured in androgen-depleted medium for 10 days, seeded at 2 × 10⁴ cells/ml and then monitored for cell growth. Cell number (mean of three biologic replicates) was determined by trypan blue staining. b Lysates from LNCaP, C4-2B and LNCaP-AI cells were immunoblotted with antibodies against the indicated proteins. c LNCaP, C4-2B and LNCaP-AI cells were analyzed for MUC1-C mRNA levels by qRT-PCR using primers listed in Supplementary Table 1. The results (mean±SD of four determinations) are expressed as relative mRNA levels compared to those obtained for LNCaP cells (assigned a value of 1)(left). Lysates were immunoblotted with antibodies against the indicated proteins (right). d LNCaP-AI cells stably expressing a tet-CshRNA or tet-MUC1shRNA were treated with vehicle or 500 ng/ml DOX for 7 days. Lysates were immunoblotted with antibodies against the indicated proteins. e LNCaP-AI/tet-CshRNA (blue circles) and LNCaP-AI/tet-MUC1shRNA (red squares) cells seeded at 2 × 10⁴ cells/ml in androgen-depleted medium were treated with vehicle (open symbols) or 500 ng/ml DOX (closed symbols) for the indicated times. Cell number (mean±SD of three replicates) was determined by trypan blue staining. f LNCaP-AI/tet-MUC1shRNA cells treated with vehicle or 500 ng/ml DOX for 7 days were assayed for invasive capacity in matrigel coated transwell chambers. Results (mean ± SD of five determinations) are expressed as the relative invasive capacity compared to that obtained with the control cells (assigned a value of 1). g LNCaP-AI/tet-CshRNA and LNCaP-AI/tet-MUC1shRNA cells seeded at 500 cells/well in six-well plates were treated with vehicle or 500 ng/ml DOX. Colonies were stained with crystal violet on day 14. The results are expressed as the colony number (mean±SD of three determinations) per well. h LNCaP-AI/tet-CshRNA and LNCaP-AI/tet-MUC1shRNA cells seeded at 5 × 10³ cells/well in ultra-low attachment six-well plates were treated with vehicle or 500 ng/ml DOX for 14 days. The results are expressed as the tumorsphere number (mean±SD of three determinations) per well. *p < 0.05 (unpaired Mann–Whitney U test). Dot plots are represented by open circles in the bar graphs. Source data are provided as a Source Data file.