Figure 5.
Influence of miR-146b-5p concentrations on the expression of the IL-12Rβ1 chain by CLL cells cocultured with CD40L-TC. (A) Purified CLL cells were transfected with the indicated miRNA mimics and cultured for 48 hours with CD40L-TC and IL-12Rβ1 mRNA concentrations determined by RT-qPCR. The cases studied were PF0024, DF0319, SR0112, CA0058, RD0468, MA0151, AR0090, LG0337, PD0164, and HG0135. Data are plotted as mean ± SD. (B) Evaluation of IL-23R and IL-12Rβ1 chain expression by flow cytometry in 1 representative CLL case (SR1-ME1077) cultured with CD40L-TC for 72 hours. Only viable cells were gated, and double-positive cells were considered IL-23R complex-positive. (C) IL-23R complex expression by CLL cells (cases PF0024, HG0135, SR0112, CA0058, DF0319, RD0468, MA0151, AR0090, PD0164, and SR1-ME1077) transfected with the indicated miRNAs and cultured with CD40L-TC for 72 hours. Data are expressed as a percentage of double (IL-12Rβ1+IL-23R+) positive cells (mean ± SD). (D) IL-12Rβ1 side chain, (E) IL-23R side chain, and (F) IL-23 side chain-only expression by the CLL cells from the same cases analyzed in (C). Data are expressed as a percentage of positive cells (mean ± SD). (G) Ki67 expression was analyzed by flow cytometry in CLL cells cultured with CD40L-TC for 72 hours following transfection of the indicated miRNA mimics. Data from a representative case (CLL RD0468) are shown. (H) Multiparametric flow cytometry test to detect cells expressing both IL-23R complex and Ki67 in the same representative experiment as in (E). The cells were stained for Ki67 and the IL-12Rβ1 and IL-23R chains of the IL-23 receptor complex. Ki67+ cells gated in (E) are highlighted by the green dots, whereas the whole cell population is indicated by gray dots. (I) Summary of Ki67+ cells determinations obtained on 11 different CLL cases (PF0024, HG0135, SR0112, CA0058, DF0319, RD0468, MA0151, AR0090, PD0164, LG0337, and SR1-ME1077) after a 72-hour culture with CD40L-TC following transfection of the indicated miRNA mimics. Data are expressed as mean ± SD. P values of the difference between treated and control cells are indicated (Wilcoxon test). ∗P < .05.

Influence of miR-146b-5p concentrations on the expression of the IL-12Rβ1 chain by CLL cells cocultured with CD40L-TC. (A) Purified CLL cells were transfected with the indicated miRNA mimics and cultured for 48 hours with CD40L-TC and IL-12Rβ1 mRNA concentrations determined by RT-qPCR. The cases studied were PF0024, DF0319, SR0112, CA0058, RD0468, MA0151, AR0090, LG0337, PD0164, and HG0135. Data are plotted as mean ± SD. (B) Evaluation of IL-23R and IL-12Rβ1 chain expression by flow cytometry in 1 representative CLL case (SR1-ME1077) cultured with CD40L-TC for 72 hours. Only viable cells were gated, and double-positive cells were considered IL-23R complex-positive. (C) IL-23R complex expression by CLL cells (cases PF0024, HG0135, SR0112, CA0058, DF0319, RD0468, MA0151, AR0090, PD0164, and SR1-ME1077) transfected with the indicated miRNAs and cultured with CD40L-TC for 72 hours. Data are expressed as a percentage of double (IL-12Rβ1+IL-23R+) positive cells (mean ± SD). (D) IL-12Rβ1 side chain, (E) IL-23R side chain, and (F) IL-23 side chain-only expression by the CLL cells from the same cases analyzed in (C). Data are expressed as a percentage of positive cells (mean ± SD). (G) Ki67 expression was analyzed by flow cytometry in CLL cells cultured with CD40L-TC for 72 hours following transfection of the indicated miRNA mimics. Data from a representative case (CLL RD0468) are shown. (H) Multiparametric flow cytometry test to detect cells expressing both IL-23R complex and Ki67 in the same representative experiment as in (E). The cells were stained for Ki67 and the IL-12Rβ1 and IL-23R chains of the IL-23 receptor complex. Ki67+ cells gated in (E) are highlighted by the green dots, whereas the whole cell population is indicated by gray dots. (I) Summary of Ki67+ cells determinations obtained on 11 different CLL cases (PF0024, HG0135, SR0112, CA0058, DF0319, RD0468, MA0151, AR0090, PD0164, LG0337, and SR1-ME1077) after a 72-hour culture with CD40L-TC following transfection of the indicated miRNA mimics. Data are expressed as mean ± SD. P values of the difference between treated and control cells are indicated (Wilcoxon test). ∗P < .05.

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