Fig. 1

CD28 activation of the PI3K/AKT signaling pathway and regulation of miR29b and DNMT3B expression in MM plasma cells (U266 and RPMI 8226 cells). Cells were cultured in the presence of 10 µg/mL anti-CD28-loaded particles, alone or with either 50 µg/mL CD28-blocking mAb (24 h). A Representative western blots of PI3K/AKT pathway components in conditions of CD28 stimulation or blocking. B Densitometric analysis of the experiment shown in (A). Band intensities were normalized to those of β-actin and the relative levels of proteins under CD28 blocking were expressed as a percentage of those during CD28 stimulation (dotted line). C Levels of miR29b and DNMT3B mRNA under conditions of CD28 blocking normalized to those of RNU44 or GAPDH, respectively, and then expressed as fold change relative to the average value for CD28-stimulated cells (data from real-time PCR and 2^−ΔΔCt method). D Representative western blot and E densitometric analysis of DNMT3B expression after CD28 stimulation or blocking. Values were normalized to β-actin and then to values for CD28-stimulated cells (dotted line). Chart data are mean and SD for 7 independent experiments. *P < .05, Wilcoxon signed-rank test