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. 2021 Jun 15;12:3622. doi: 10.1038/s41467-021-23330-6

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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. 5 — a Proliferation of human cytotoxic T lymphocytes. Human donor blood CD8 + lymphocytes were not activated (Non-Act) or activated by incubation with CD3/28 beads (Act.) and cultured alone (NT), or coincubated with isolated human donor blood neutrophils pretreated for 6 h with vehicle (PBN) or 5 μM GSK2830371 (PBN + GSK) (n = 4 each condition). b Proliferation of murine cytotoxic T lymphocytes. Ppm1d^+/+ peripheral blood Cd8+ lymphocytes were not activated (NT) or activated by incubation on CD3/28-coated plates (Act.) and cultured alone (NT), coincubated with Ppm1d^+/+ neutrophils pretreated for 6 h with vehicle (WT PBN) or 5 μM GSK2830371 (WT PBN + GSK), or coincubated with Ppm1d^KO2/KO2 neutrophils (KO PBN) (n = 4 each condition). c Expression of 4-1BBL mRNA (left panel) and OX40L mRNA (right panel) in neutrophils isolated from B16 tumors engrafted in Ppm1d^+/+ mice (WT TAN) or Ppm1d^KO2/KO2 mice (KO TAN), isolated from PB of Ppm1d^+/+ mice and treated for with vehicle (WT PBN) or 5 μM GSK2830371 (WT PBN + GSK), or isolated from PB of Ppm1d ^KO2/KO2 mice (KO PBN) (n = 3 each condition). d Luciferase reporter assay of HCT116 p53^+/+ and HCT116 p53^−/− cells transfected with pGL3 vector expressing luciferase under regulation of the human TNFSF9 (4-1BBL) promoter (left panel, n = 8) or the human TNFSF4 (OX40 L) promoter (right panel, n = 4). e Relative levels of 4-1BBL mRNA (left panel) and OX40L mRNA (right panel) in PBN isolated from Ppm1d^+/+ mice (WT), Ppm1d^KO2/KO2 mice (KO), or Ppm1d^KO2/KO2/Trp53^KO/KO double-knockout mice (DKO) (n = 4 each genotype). f Tumor volume (left) and AUC (right) for growth of B16 F10 tumors in Ppm1d^fl/fl (WT), Ppm1d^KO2/KO2 (KO2), or Trp53/Ppm1d double-knockout (DKO) mice (n = 4 each genotype). g Expression of 4-1BBL (left panel) and OX40L (right panel) protein levels on the surface of PPM1D^KO2/KO2 neutrophils isolated from bone marrow, spleen, and blood (n = 8 each, except LysM-Cre BM n = 6). h Tumor volume for growth of B16 F10 tumors in Ppm1d^LysM-Cre mice after inactivation of 4-1BBL and OX-40L ligands on the surface of cells with serial injection of neutralizing anti-4-1BBL and anti-OX40L antibodies (n = 4 for each group, except Ppm1d^fl/fl + α-IgG ctrl n = 7). Data are depicted as means ± SEM. Student’s unpaired t test (two-tailed) (panel d) and Mann–Whitney’s test (two-tailed) (panel g), one-way ANOVA (panels a–c, e, f), or two-way ANOVA (h): *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001 (one representative experiment out of three is shown for panels f and h). Source data are provided as a Source Excel Data file.