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. 2018 Nov 30;9:5108. doi: 10.1038/s41467-018-07470-w

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© The Author(s) 2018

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. 6 — EC-Trp53 deletion attenuates endothelial CXCR4 expression and M2 polarization of SDF-1⁺ TAMs after a single 20-Gy irradiation. a RT-qPCR analysis of CXCR4 in HUVECs 48 h post 10 Gy irradiation. HUVECs were transfected with TGFβR2, Trp53, or TGFβR2+Trp53 siRNAs 2 days before irradiation. si-TGFβR2 + IR or si-Control + IR versus si-Control group, ####p < 0.0001.si-p53 + IR or si-TGFβR2+si-p53 + IR versus si-Control + IR or si-TGFβR2 + IR group, respectively, ***p < 0.001 and ****p < 0.0001. b Immunofluorescence of CD31 and CXCR4 in KP tumours. CXCR4⁺CD31⁺ cell quantitation (magnification, ×200) is shown. Scale bar = 20 μm. c Immunofluorescence of SDF-1, F4/80, and CD206 in KP tumours, with or without irradiation (23 dpi). Quantification of SDF-1⁺ cells (magnification, ×200) is shown. Scale bar = 50 μm. d Immunofluorescence of SDF-1 and CD31 in KP tumours from WT mice 1 dpi. Scale bar = 10 μm e Immunofluorescence of F4/80, SDF-1, and CD206 in KP tumours 7 dpi. Scale bar = 20 μm (crop, 5 μm). Quantification of CD206⁺ and iNOS⁺ cells among SDF-1⁺F4/80⁺ cells (magnification, ×200). f Flow-cytometric analysis of SDF-1⁺ cells in CD206⁺F4/80⁺ cells of KP tumours at 7 dpi. g Immunofluorescence of F4/80, SDF-1, and CD206 in BMDMs after a 48-h coculture with KP tumour-derived ECs. As controls, BMDMs from WT mice were polarized to M1 or M2 macrophages by treatment with LPS (100 ng/ml) plus IFNγ (20 ng/ml), or IL-10 (20 ng/ml) plus IL-4 (20 ng/ml), respectively, for 48 h. Scale bar = 20 μm. h Immunofluorescence of CD31, SDF-1, and CD206 in KP tumours from WT mice at 7 dpi, with or without AMD3100 treatment. BrdU was used for in vivo proliferation assays. CD206⁺SDF-1⁺ TAMs, iNOS⁺SDF-1⁺ TAMs, and non-differentiated SDF-1⁺ monocytes are marked with yellow, green, and open arrows, respectively. Quantification of CD206⁺SDF-1⁺ and iNOS⁺SDF-1⁺ cells among F4/80⁺ cells (magnification, ×200) and flow-cytometric analysis of BrdU⁺SDF-1⁺ cells in F4/80⁺ cells. Scale bar = 10 μm. i Flow-cytometric analysis of SDF-1⁺CD206⁺ cells in F4/80⁺ cells of WT BMDMs after coculture with KP tumour-derived ECs from WT and EC-p53KO mice for 48 hpi, with or without AMD3100 treatment. Error bars indicate SD (a) or SEM (c, e, f, h, i); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant (one-way ANOVA). Data are representative of three independent experiments