TLR9 stimulation induces p53 expression.

<p><b>(A)</b> Normal B-cells were treated with CpG-ODN (0.5 μg/ml) in the presence or absence of RA (200 nM) for 24 hours prior to irradiation (IR; 10 Gy). After indicated time points B-cells were collected and subjected to western blot analysis with antibodies recognizing calnexin (as loading control), p53, or p53 phosphorylated at serine 15 (S15). The right panel shows a more exposed version of the blot depicting induction of p53 in non-irradiated cells. One representative experiment of three is shown. Original uncropped blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185708#pone.0185708.s005" target="_blank">S5 Fig</a>. <b>(B)</b> Densitometric analysis of the p53 expression (based on low exposure) at 4 hours after irradiation was normalized to calnexin. The results are presented as histograms of mean values ±SEM (n = 6, *<i>p</i> < 0.05, **<i>p</i> < 0.01, paired t-test). <b>(C)</b> B-cells were treated as described in (A), and lysates were subjected to western blot analysis with antibodies recognizing calnexin (as loading control) and p21^Cip. One representative experiment of three is shown. Original uncropped blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185708#pone.0185708.s006" target="_blank">S6 Fig</a>. <b>(D)</b> B-cells were stimulated as described in (A), and the cells were harvested 24 hours after irradiation. The mRNA level of <i>TP53</i> was quantified by RT-qPCR. The amounts of <i>TP53</i> mRNA related to reference genes (<i>TBP</i> and <i>B2M</i>) were quantified using the 2^-ΔCt-method. The results are presented as histograms of mean values ±SEM (n = 4 *<i>p</i> < 0.05, paired t-test).