10.1371/journal.pgen.1001360.g001 Daniel Menendez Daniel Menendez Maria Shatz Maria Shatz Kathleen Azzam Kathleen Azzam Stavros Garantziotis Stavros Garantziotis Michael B. Fessler Michael B. Fessler Michael A. Resnick Michael A. Resnick Induced expression of the <i>TLR</i> gene family in primary human T-lymphocytes by DNA stressors and activation of the p53 pathway in cells from healthy subjects. Public Library of Science 2011 t-lymphocytes dna stressors activation p53 pathway cells 2011-03-31 02:09:45 Figure https://plos.figshare.com/articles/figure/_Induced_expression_of_the_TLR_gene_family_in_primary_human_T_lymphocytes_by_DNA_stressors_and_activation_of_the_p53_pathway_in_cells_from_healthy_subjects_/457785 <p>Human peripheral blood mononuclear cells (PBMC) freshly isolated from healthy subjects (n = 17–18) were incubated with PHA to stimulate T-lymphocyte expansion. After 48 h incubation, cells were exposed to (A) ionizing radiation (IR); (B) 5-fluorouracil (5FU); (C) doxorubicin or (D) nutlin. Cells were harvested following 24 h treatment in the presence of PHA. In the case of IR, they were harvested 24 h after exposure to 4 Gy. (Robust responses were observed under these conditions; however, further studies might reveal more optimum times and dose for primary cells.) Presented in panels A to D are mRNA expression levels of <i>TLRs</i> and <i>p21<sup>WAF1</sup></i> as compared to expression in untreated cells for each subject (the dashed red line corresponds to no change with treatment, <i>i.e.</i>, a value of 1; horizontal bars correspond to the median). Gene expression was analyzed by qPCR and normalized to 18S ribosomal RNA. <i>Statistical analysis in A–D</i>. Unless noted, the <i>TLR</i> genes of the population as a whole exhibited statistically significant change in expression induced by an agent (vs. untreated) at the p<0.006 level (see text). Black arrows indicate changes that are not significantly different from the control for specific <i>TLR</i> genes in the population (p≥0.05). (E) Changes in <i>TLR</i> expression after IR of cells from all subjects (BS#) presented as a heat map. The subjects are grouped according to IR responsiveness (<i>i.e.</i>, purple, for nearly all <i>TLR</i>s being inducible and blue for people with only a few <i>TLR</i>s induced by IR). (F) p53 and p21 activation analyzed by Western blot in a representative experiment for subject BS#7 with actin as a loading control. (G) Inhibition of IR-induced <i>TLR</i> gene expression by the p53 inhibitor pifithrin-α (PFTα, 40 µM) added to cells 2 h prior to IR and kept there after exposure. Values are relative to cells treated with DMSO represented as the dash red line, <i>i.e.</i>, a value of 1. Presented is a representative experiment with T-lymphocytes from subject BS#19.</p>