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. 2023 Jul 27;14:4524. doi: 10.1038/s41467-023-40170-8

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© The Author(s) 2023

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/.

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Fig. 7 — a–f AT3-OVA mammary tumor cells were injected into the fourth inguinal mammary fat pads of 8 week-old Ptpn2^fl/fl (n = 6) and Lck-Cre;Ptpn2^fl/fl (C57BL/6) (n = 5-6 in each group) female mice. Mice were treated with Compound 182 (CMP-182; 10 mg/kg i.v.; n = 6) or saline (n = 6) on days (d) 7, 9, 11, 13, 15, 17, 19 and 21 after tumor cell implantation. a Tumor growth was monitored and b tumor weights were measured. c Tumor-infiltrating lymphocytes including CD44^hiCD62L^lo CD8⁺ and CD4⁺ effector/memory (EM) T cells, CD19⁺ B cells, NK1.1⁺TCRβ⁻ (NK) cells, CD4⁺CD25⁺FoxP3⁺ regulatory T cells (T_regs) and granulocytic and monocytic CD11b⁺F4/80^hi/loLy6C⁺Ly6G^+/−myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry. d Tumor-infiltrating T cells from (a) were stimulated with PMA/Ionomycin in the presence of Golgi Stop/Plug and stained for intracellular IFN-γ and TNF. Intracellular granzyme B (GRZMB) was detected in unstimulated tumor-infiltrating CD8⁺ T cells. AT3-OVA tumors were processed for e immunohistochemistry staining for p-STAT-1, or CD3 (counterstained with hematoxylin) or f qPCR monitoring for the expression of STAT-1 target genes. g AT3-OVA mammary tumor cells were injected into the fourth inguinal mammary fat pads of C57BL/6 mice. Mice were treated with CMP-182 (10 mg/kg i.v.) or saline on days 6, 8, 10, 12, 14, 16, 18, and 21 after tumor cell implantation; the resultant tumor growth curves are shown in Fig. 4a. The resulting AT3-OVA tumors were processed for immunohistochemistry staining for STAT-1 Y701 phosphorylation (p-STAT-1), or CD3 (counterstained with hematoxylin). In (a–d, f) representative results (means ± SEM) from at least two independent experiments are shown. In (e, g) micrographs are representative of two independent experiments with 5 mice per group. Significance for tumor sizes in (a) was determined using a 2-way ANOVA Test and for tumor weights in (b) using a 1-way ANOVA Test. In (c, d) significances were determined using a 1-way ANOVA Test and a 2-tailed Mann–Whitney U Test (^#p < 0.05, ^##p < 0.01) where indicated. In (f) significances were determined using a 1-way ANOVA Test.