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. 2017 Jan 13;18(1):145. doi: 10.3390/ijms18010145

Figure 4.

Figure 4

Relationship between the expression of miR-34a-5p and that of the miR-34a-5p targets during the HPCs commitment. (A) Detection of lymphoid enhancer-binding factor 1 (LEF1), nuclear receptor subfamily 4, group A, member 2 (NR4A2) and MYB expression levels by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) in Negative Control (NegCTR) mimic and miR-34a-5p mimic-transfected CD34+ cells at 24 h post-nucleofection; (B) Western Blotting analysis of Lef1 (i), Nr4a2 (ii) and Myb (iii) protein levels in protein lysates from miR-34a-5p mimic-transfected compared to NegCTR mimic-transfected CD34+ cells at 24 h after the last of two nucleofection cycles. Actin protein levels are reported as loading control; (C–F) Expression kinetics of miR34a (i), LEF1 (ii) and NR4A2 (iii) during the megakaryocyte (C); monocyte/macrophage (D); erythroid (E) and granulocyte (F) differentiation of CD34+ cells. miR-34a-5p, LEF1 and NR4A2 expression levels were monitored by RT-qPCR at day 4 and day 8 post-CD34+ cells purification. Modulations of miR-34a-5p, LEF1 and NR4A2 transcripts are reported as Relative Quantity (RQ) respect to freshly purified CD34+ cells (DAY 0) sample, which was set as calibrator. Data are from n = 3 independent experiments performed with different healthy donor-derived cord blood units. Values in the graph are reported as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 compared to freshly purified CD34+ cells (DAY 0) sample.