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. 2021 Aug 20;6:313. doi: 10.1038/s41392-021-00730-0

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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. 6 — Inhibition of BCL9 promotes CD155-CD226 checkpoint, which signals via VAV1 phosphorylation. a Percentage of CD96⁺ cells among CD45⁺CD8⁺ T cells from MC38 tumor tissue in BCL9^+/+ and BCL9^−/− mice was analyzed. b qRT-PCR measurement of Cd96 in CD8⁺ T cells treated with NT-shRNA or Bcl9-shRNA. c Percentage of CD226⁺ cells among CD45⁺CD8⁺ T cells from CT26 tumor tissue in BALB/c mice inoculated with wild-type (WT), NT-shRNA or Bcl9-shRNA-transduced-CT26 cells was analyzed. d Percentage of CD226⁺ cells among CD45⁺CD8⁺ T cells from MC38 tumor tissue in C57BL/6 mice inoculated with WT, NT-shRNA or Bcl9-shRNA-transduced-MC38 cells was analyzed. e Percentage of Ki67⁺ cells among CD45⁺CD8⁺ T cells from MC38 tumor tissue in C57BL/6 mice inoculated WT, NT-shRNA or Bcl9-shRNA-transduced-MC38 cells was analyzed. f Proliferation of CD8⁺ T cell co-cultured with CT26 were treated with Bcl9-shRNA or hsBCL9_CT-24 and analyzed by Cell titer-Glo^® Luminescent cell viability assay. g Mouse CD8⁺ T cells proliferation was measured after treatment with NT-shRNA or Bcl9-shRNA in the presence of anti-CD155 and anti-CD226 antibodies. h Immunoblots showing VAV1 and VAV1 phosphorylation on Tyr174 (p-VAV1) in equal amounts of total lysates from CD8⁺ T cells that were treated with or without CD3 antibody (Thermo, 16-0032-81, 0.25 μg) and/or anti-CD226 antibody (Thermo, 0.25 μg) for 5 min. i Immunoblots showing phosphorylated VAV1 (p-VAV1), phosphorylated ERK1/2 (p-ERK1/2), phosphorylated AKT (p-AKT) as well as total VAV1, AKT, and ERK1/2 in equal amounts of total lysates from CD8⁺ T cells treated with NT-shRNA or Bcl9-shRNA. j qRT-PCR measurement of CD155/Pvr in CT26 cells treated with hsBCL9_CT-24 (5 μM) or vehicle for 24 h. k qRT-PCR measurement of CD155/Pvr expression in CT26 cells transduced with NT-shRNA or Bcl9-shRNA. l qRT-PCR measurement of Gli1 expression in CT26 cells treated with NT-shRNA or Bcl9-shRNA. m qRT-PCR measurement of Ptch1 expression in CT26 cells treated with NT-shRNA or Bcl9-shRNA. Results were denoted as means ± SEM for experiments performed in triplicate. Each experiment was repeated three times, and the statistical significance of differences between groups was determined by non-parametric test. P < 0.05 means statistically significant.