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. 2015 May 11;6(21):18484–18503. doi: 10.18632/oncotarget.3941

Figure 7. CpG/CD40L/c-derived CLL B-cells differentiation induces downregulation of the expression of CLL-pathogenesis-associated factors, decreased survival and a low proliferation rate.

Figure 7

On D0, CLL B-cells were stimulated with CpG and CD40L, in combination with the cytokines IL-2, IL-10 and IL-15. On D4, cells were harvested and incubated with IL-2, IL-6, IL-10 and IL-15 for 3 days. A. The expression of the IRF4, PRDM1, LEF1, TCL1, ROR1, FMOD, TNFRSF13B, BIRC5 and Ly9 genes was evaluated by quantitative real-time RT-PCR on D0 and D7. Results are expressed relative to gene expression in CLL B-cells on D0, according to the 2−ΔΔCT method. The results are represented as log2 fold changes in box-and-whisker (min to max) plots (the “+” sign indicates the mean) for seven experiments. Statistical significance was calculated using the Wilcoxon's test: *p < 0.05, ns, not significant. B. Cells were stained with Annexin V-PE and 7AAD at D0 and D7 to evaluate apoptosis. Left panel: the percentages of double-negative (i.e. annexin-V-negative and 7AAD-negative) living cells in seven experiments are represented in box-and-whisker (min to max) plots (the “+” sign indicates the mean). Right panel: cytometry plots from a representative patient. Statistical significance was calculated using the Wilcoxon's test: *p < 0.05. C. Immunoblot analysis and densitometry values of BLIMP1, BCL2, BCLxL, and PUMA in cells at D0 and D7. The PUMA antibody also cross-reacts with an 18 kDa band of unknown origin. The black dividing lines on the blot data indicate that lanes are run on different parts of the same gel (non-adjacent lanes). The data shown are representative of three experiments. Statistical significance was calculated using Student's t-test: *p < 0.05. D. At D0 and D7 of culture, the DNA content of living cells was measured by DyeCycle Violet staining. Results are represented as the summed percentages of cells in the S and G2/M phases of the cell cycle. Cytometry plots are representative of the results from five experiments. Day 7 values were compared with D0 values and statistical significance was calculated using Wilcoxon's test: *p < 0.05.