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. 2018 Jan 30;32(4):1003–1015. doi: 10.1038/leu.2017.336

Figure 3.

Figure 3

(a) Representative flow cytometry analysis of CD86 and CD83 expression on DCs after miR-29b transient transfection and 48 h co-culture with U266 cell lines or with maturation stimuli (lipopolysaccharide). In both cases the percentage of mature DCs is decreased by the enforced expression of miR-29b. (b) Evaluation of cytokines production and secretion in the supernatant of DCs after miR-29b or NC transfection and 48 h co-culture with MM cells. Plots represent mean and s.d. of six different experiments. *P<0.05. (c) Migration assay to evaluate changes in the capability to attract CCR2+ and/or CCR6+ inflammatory cell populations from PBMCs, between supernatant of 29b-DCs and NC-DCs co-cultured for 48 h with three different MM cell lines (U266, RPMI8226 and MM1S). Plots represent mean and s.d. of three different experiments. *P<0.05. (d) Western blot evaluation of the main signaling pathways involved in inflammatory response (NFκB, STAT3, mitogen-activated protein kinase, JUN and suppressor of cytokine signaling 1 (SOCS1)) in DCs after miR-29b transfection and 48 h co-culture with MM cells. (e) Results from tubulogenic assay performed in the presence of supernatant from 29b-DCs or NC-DCs co-cultured with RPMI8226 MM cells. Images have been analyzed with ImageJ software and Angiogenesis analyzer plugin. The histograms under the pictures represents the estimation of tubulogenic potential obtained by analyzing the number of nodes (pixels with at least three neighboring elements corresponding to a bifurcation), segments (elements delimited by two junctions), meshes (areas enclosed by segments or master segments, made by tube-like-structures) number and total area. Legend: red points surrounded by blue, nodes surrounded by junctions symbol; red surrounded by yellow, extremities; green, branches (elements constituted by a junction and one extremity); magenta, segments; orange, master segments (segments where none of the two junctions is implicated with one branch); blue sky, meshes; junctions surrounded by red, master junctions (junctions linking at least three master segments); blue and cyan, isolated elements. *P<0.05. (f) Evaluation of the AKT signaling (pAKT, AKT and phosphatase and tensin homolog (PTEN)) through western blotting, accompanied by the demonstration of PTEN downregulation at the mRNA level and validation of PTEN as a miR-29b target through luciferase reporter assay. *P<0.05.