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. 2020 Feb 28;11:143. doi: 10.3389/fphar.2020.00143

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Copyright © 2020 Chimote, Gawali, Newton, Wise-Draper and Conforti

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Potentiation of chemotaxis in the presence of adenosine and INFγ production by 1-EBIO in CALM1-knockdown HD T cells. (A) Y-COM values calculated for HD T cells transfected with siCALM1 migrating along either a CXCL12 gradient or a combination gradient of CXCL12 with adenosine (ADO) with or without preincubation with 20 μM 1-EBIO (n = 4 donors). Transfected cells, identified by positive GFP staining, were tracked for analyzing Y-COM. (B) Percentage inhibition by ADO of Y-COM (B) in cells pretreated with 1-EBIO. Horizontal red line represents mean values for each group. (C) (Left) Representative flow cytometry plots gated on CD3⁺ T cells transfected with either scr-RNA or siCALM1 with or without 1-EBIO (100 μM) showing IFNγ production after stimulation. Transfected cells were identified by positive staining for GFP. (Right) Quantification of IFNγ induction in scr-RNA or siCALM1 transfected CD3⁺ T cells with or without 1-EBIO in three HD. The data were normalized to IFNγ production in control scr-RNA transfected HD cells. Bars represent mean ± SEM. (D) (Left) Representative flow cytometry plots gated on HD or HNSCC CD3⁺ T cells with or without 1-EBIO (100 μM) showing IFNγ production after stimulation. (Right) Quantification of IFNγ-producing HD and HNSCC T cells after stimulation in the presence or absence of 1-EBIO. The data were normalized to IFNγ production in control HD cells. For the HD + 1-EBIO condition, bars represent mean ± range for two individuals, while the rest of the bars in the graph represent mean ± SD for data from three HD and HNSCC patients. For analysis of (C, D), live cell population was identified by exclusion from Zombie Aqua live/dead stain and gated on CD3⁺ population and transfected cells were identified by positive staining for GFP (in panel C). IFNγ⁺ T cell populations were selected by drawing rectangle gates. The numbers in the gates are the percentages of IFNγ⁺ CD3⁺ T cells. Data in (A) were analyzed with one-way repeated measures ANOVA (p <0.001), in (B) with paired Student's t test, and in (C) with a two-way ANOVA [p = 0.207 for scr-RNA vs. siCALM1 and p = 0.006 for – 1-EBIO vs. +1-EBIO]. Data in (D) were analyzed by Kruksal-Wallis One Way ANOVA on Ranks (p < 0.001).