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. 2025 Dec 19;17:761. doi: 10.1038/s41467-025-67437-6

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© The Author(s) 2025

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

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Fig. 4 — a, b The representative image and corresponding quantification of flow cytometry analysis of M1 (CD86⁺) polarization in total murine macrophages (RAW264.7 cells) without any treatments (Blank), with only LPS/IFN-γ stimulation (Ctrl), with LPS/IFN-γ stimulation coupled by pure reuterin (Reuterin) or MnL/Gly treatments (n = 3). c, d The representative image and corresponding quantification of flow cytometry analysis of M2 (CD206⁺) polarization in total macrophages (RAW264.7 cells) without any treatments (Blank), with only IL-4 stimulation (Ctrl), with IL-4 stimulation coupled by reuterin or MnLR/Gly treatments (n = 3). e Images of the 3D MCTs with different treatments at the given time points. The area within the red circle showing the tumor spheroids’ morphology. Scale bar, 25 μm. f, g The representative image and corresponding quantification of flow cytometry analysis of the ratio of M1 (CD86⁺) to M2 (CD206⁺) macrophages in CD45⁺CD68⁺ hPBMC-macrophages after reuterin or MnLR/Gly treatments (n = 3). h The levels of immune functional cytokines (IL-6, TNF) produced by hPBMC-macrophages after the given treatments (n = 3). i, j The representative images and corresponding quantification of flow cytometry of the proportion of CFDA-SE⁺ cells (DLD-1) in CD11b⁺ hPBMC-macrophages after being stimulated by the given agents (phagocytic function assessment) (n = 4). k, l Immunoblotting assays of HIF-1α protein within the RAW264.7 cells with the given treatments. Three times were repeated independently with similar results. The representative image and corresponding quantification of flow cytometry analysis of M1 (m, n) or M2 (o, p) polarization in the total LPS/IFN-γ-stimulated or IL-4-stimulated RAW264.7 cells with the given treatments (n = 3). q Immunoblotting assays of HIF-1α protein within the hPBMC-macrophages with the given treatments. Three times were repeated independently with similar results. r, s The representative image and corresponding quantification of flow cytometry analysis of the ratio of M1 (CD86⁺) to M2 (CD206⁺) macrophages in CD45⁺CD68⁺ hPBMC-macrophages with the given treatments (n = 3). iHIF-1α, HIF-1α inhibitor (YC-1). t The schematic illustration of the impact of MnLR/Gly on macrophage polarization. The “n” represents the number of biologically independent samples. Data are shown as mean ± SD; p-value (compared to Ctrl in g, h, j); one-way ANOVA (one-tailed), Tukey’s multiple comparisons. Source data are provided as a Source Data file.