Figure 2.

Effects of miR-486-3p overexpression, miR-486-3p knockdown and miR-486-3p rescue upon MYB silencing on megakaryocyte differentiation. (a) Flow cytometric analysis (mean±S.E.M.; n=4) of CD34, CD41 and CD42b expression at day 4 of megakaryocyte unilineage culture post nucleofection. (b) Flow cytometric analysis (mean±S.E.M.; n=4) of CD41 and CD42b expression at day 12 of megakaryocyte unilineage culture post nucleofection. (c) Morphological analysis of NegCTR mimic (i) and miR-486-3p mimic-transfected cells (ii) after May–Grünwald–Giemsa staining at day 8 of megakaryocyte unilineage culture post nucleofection in a representative experiment. Magnification, × 1000. (d) Culture micrographs of NegCTR mimic (i) and miR-486-3p mimic-transfected cells (ii) at day 11 of megakaryocyte culture at × 200 magnification in a representative experiment. NegCTR mimic sample (i) shows the large, polyploid cells together with several proplatelet-forming (see black arrows) cells, which are almost undetectable in miR-486-3p-overexpressing sample (ii). (e) Megakaryocyte clonogenic assay results (mean±S.E.M.; n=4). CFU-MKs were scored according to the manufacturer's protocol based on their size, which reflects the maturation stage of the progenitor giving rise to each colony, as large (>50 cells, arising from more primitive MK progenitors), medium (21–49 cells) and small (3–21 cells, deriving from more mature megakaryocyte progenitors) colonies. The cells were plated 24 hours after the last nucleofection and scored after 12 days. Values are reported as number of megakaryocyte colonies for 4000 plated cells. *P≤0.05, **P≤0.01 and ***P≤0.001 in miR-486-3p mimic compared with NegCTR mimic sample. The efficient upregulation of miR-486-3p expression upon mimic transfection was checked at 24 hours post nucleofection by qRT-PCR (RQ±SE, 74.4±19.2, P<0.05) (f–h) Flow cytometric analysis (mean±S.E.M.; n=3) of CD34, CD41 and CD42b expression at day 4 (f) and CD41 and CD42b at day 8 (g) and day 12 (h) of megakaryocyte unilineage culture post nucleofection with miR-486-3p/NegCTR inhibitor. (i) Megakaryocyte clonogenic assay results (mean±S.E.M.; n=3) for miR-486-3p and NegCTR Inhibitor-transfected CD34+ cells. CFU-MKs were scored according to the manufacturer's protocol based on their size, as reported above. Values are reported as number of megakaryocyte colonies for 4000 plated cells. *P≤0.05 in miR-486-3p inhibitor compared with NegCTR-inhibitor sample. The efficient knockdown of miR-486-3p expression upon inhibitor transfection was checked at 24 hours post nucleofection by qRT-PCR (RQ±SE, 0.593±0.161, P<0.05) (j and k) Flow cytometric analysis (mean±S.E.M.; n=3) of CD34, CD41 and CD42b expression at day 4 (j) and CD41 and CD42b at day 12 (k) of megakaryocyte unilineage culture post nucleofection with MYBsiRNA/NegCTRsiRNA and/or miR-486-3p/NegCTR mimics. (l) Megakaryocyte clonogenic assay results (mean±S.E.M.; n=3) for CD34+ cells transfected with MYBsiRNA/NegCTRsiRNA and/or miR-486-3p/NegCTR mimic. CFU-MKs were scored according to the manufacturer's protocol based on their size, as reported above. Values are reported as number of megakaryocyte colonies for 4000 plated cells. *P≤0.05, **P≤0.01 and ***P≤0.001. MYB and miR-486-3p expression levels were checked at 24 hours post nucleofection by qRT-PCR (RQ±SE, 0.423±0.122 in MYBsiRNA/NegCTR mimic and 0.487±0.148 in MYBsiRNA/miR-486-3p mimic for MYB; 75.821±13.347 in NegCTRsiRNA/miR-486-3p mimic and 70.382±11.027 in MYBsiRNA/miR-486-3p mimic for miR-486-3p levels; NegCTRsiRNA/NegCTR mimic set as calibrator). Abbreviations: CFU, colony forming unit; MK, megakaryocyte; MYBsiRNA, MYB-targeting siRNA; NegCTR inhibitor, negative control inhibitor; NegCTR mimic, negative control mimic; NegCTRsiRNA, negative control siRNA; and n=number of experiments