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. 2021 Dec 3;41(6):865–877. doi: 10.1038/s41388-021-02133-5

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© The Author(s) 2021

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. 3 — a GSEA of myeloid cell activation signature in PD-L1^hi PDAC samples vs. PD-L1^lo counterparts from the TCGA dataset. b TCGA analysis between the scoring of macrophage signature and PKM2 (left) or PD-L1 (middle). Also, correlation between M2 macrophage signature and PD-L1 was calculated in PDAC (right). c Representative immunofluorescence staining of CD68⁺ macrophages (green), PD-L1+ cells (red), and PKM2+ cells (white) in PDAC tissues with FDG^hi or FDG^lo. Scale bar, 20 μm. d Representative immunostaining of CD163⁺/CD206⁺ macrophages from paired tumors of patients with PDAC with FDG^hi or FDG^lo. Red arrowheads showed CD163⁺ or CD206⁺ macrophages. Scale bar, 50 μm. e BxPC-3/Capan-2 cells were co-cultured with M0, M1, or M2 polarized macrophages (tumor cell:macrophage = 1:2) for 48 h. Then, BxPC-3/Capan-2 cells were lysed and assayed for the expression of PD-L1 and PKM2 mRNA by qRT-PCR. f PKM2 and PD-L1 protein expression levels from whole-cell lysate of BxPC-3/Capan-2 cells co-cultured with M0/M2 macrophages were determined by western blotting. g BxPC-3/Capan-2 cells co-cultured with M0/M2 macrophages were cross-linked by glutaraldehyde first and then subjected to western blotting. h Nuclear and cytosolic lysates were prepared from BxPC-3/Capan-2 cells co-cultured with M0/M2 macrophages, followed by western blotting. i Subcellular localization of PKM2 in BxPC-3/Capan-2 cells co-cultured with M0/M2 macrophages. Cells were immunostained with anti-PKM2 (PKM2, red). The nucleus was marked with 4′,6-diamidino-2-phenylindole dihydrochloride (blue). j BxPC-3/Capan-2 cells were pretreated with TEPP-46 (10 μM) for 12 h or/and then co-cultured with M2 polarized macrophages (tumor cell:macrophage = 1:2) for 48 h. Then, PKM2 and PD-L1 protein expression levels from whole-cell lysate of BxPC-3/Capan-2 cells were determined by western blotting. k BxPC-3/Capan-2 cells pretreated with TEPP-46 or/and co-cultured with M2 macrophages were cross-linked by glutaraldehyde first and then subjected to western blotting. l Nuclear and cytosolic lysates were prepared from BxPC-3/Capan-2 cells pretreated with TEPP-46 or/and co-cultured with M2 macrophages, followed by western blotting. Data were shown as mean ± SEM from three experiments. Statistical significance was determined by a t-test; ^*P < 0.05,^**P < 0.01,^***P < 0.001. NS = no significance.