Induction of UCK2 expression through the activation of the TCR signaling pathway. (A) CD4⁺ T cells isolated from 5 healthy volunteers were cultured in the presence (blue line) or absence (red line) of anti-CD3/CD28 antibodies conjugated beads. At 1, 3, and 5 days after seeding, cells were stained with trypan blue, and the number of live cells was counted. The results represent the mean of 5 biological replicates of CD4⁺ T cells isolated from 5 independent donors. (B) Real-time PCR conducted to measure expression of the UCK2 and UCK1 genes by T cells treated (or not) with anti-CD3/CD28 antibody-conjugated beads for 1 day. Graphs show relative expression of the UCK2 and UCK1 genes (compared with untreated T cells) after normalization to ACTB mRNA levels. The results are expressed as the mean ± standard deviation (SD) of 3 biological replicates. (C) Immunoblots showing upregulation of UCK2 expression 5 days after treatment with anti-CD3/CD28 antibody-conjugated beads. Although 5 biological replicates of T cells isolated from 5 different donors were prepared, untreated samples were pooled, and analyzed as a single sample because the cells were small and did not grow. (D) The graph shows relative expression relative to that in a pooled untreated sample. Results are expressed as the mean ± SD of 5 biological replicates of CD4⁺ T cells isolated from 5 independent donors. (E) Expression of UCK2 and UCK1 genes in TL-Om1 cells treated for 16 hours with DHMEQ. PCR, polymerase chain reaction.