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. 2024 Jun 27;43:180. doi: 10.1186/s13046-024-03101-z

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Shikonin inhibits CAF-stimulated metastatic potential of TNBC cells. (A) Cell migration ability was measured by the wound-healing assay. TNBC cells (stained with CellTracker™ Green, green fluorescence) were co-cultured with CAFs at a ratio of 2:1 and treated with or without shikonin (2 μM) for 48 h. Images were captured at 0 and 48 h post-wounding (magnification, ×50; scale bars, 200 μm, n = 3). (B) Cell migration was measured by wound-healing assay. TNBC cells were treated with CAF-CM in the presence or absence of shikonin (2 μM) for 48 h. Images were captured at 0 and 48 h post-wounding (magnification, ×50; scale bars, 200 μm, n = 3). (C) Cell invasion was measured by Transwell assay. TNBC cells were pretreated with shikonin (2 μM) for 6 h and then inoculated in the upper compartment, and CAFs were inoculated in the lower compartment. TNBC cells and CAFs were cocultured for 24 h and images were captured (magnification, ×100; scale bars, 200 μm, n = 3). (D) Cell invasion was measured by Transwell assay. TNBC cells were treated with CAF-CM in the presence or absence of shikonin (2 μM) for 48 h. Images were captured (magnification, ×100; scale bars, 200 μm, n = 3). (E) TNBC cells grown in Matrigel. TNBC cells were treated with CAF-CM in the presence or absence of shikonin (2 μM) for 3 days (magnification, ×50; scale bars, 200 μm). (F) Immunofluorescence staining of TNBC cells labeled with anti-EpCAM antibody (red fluorescence), F-actin (green fluorescence), and DAPI (blue fluorescence). TNBC cells were co-cultured with CAFs at a ratio of 2:1 and treated with or without shikonin (2 μM) for 48 h. Arrows indicate filopodia and lamellipodia (magnification, ×400; scale bars, 50 μm). Cells were treated with or without shikonin (2 μM) in the presence or absence of CAF-CM for 48 h. (G, H) Anoikis of TNBC cells was detected by Annexin V-fluorescein isothiocyanate/propidium iodide (PI) double staining (G; n = 3) or calcein AM/ethidium homodimer (EthD-1) staining (H; magnification, ×200; scale bars, 100 μm; n = 10). Data are presented as mean ± SD. ^**p < 0.01