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. 2015 Apr 10;22(12):1906–1921. doi: 10.1038/cdd.2015.30

Figure 3.

Figure 3

Effects of miR-486-3p overexpression, miR-486-3p knockdown and miR-486-3p rescue upon MYB silencing on erythroid differentiation. (a) Flow cytometric detection (mean±S.E.M.; n=4) of CD36 and GPA (intermediate and late erythroid markers, respectively) at days 4, 8 and 12 of erythroid unilineage culture post nucleofection. *P≤0.05 and **P≤0.01 in miR-486-3p mimic compared with NegCTR mimic sample. (b) Morphological analysis of NegCTR (i) and miR-486-3p mimic-transfected cells (ii) after May–Grünwald–Giemsa staining at day 8 of erythroid unilineage culture post nucleofection in a representative experiment. Magnification, × 400. NegCTR mimic sample (i) displays more immature cells such as proerythroblasts and basophilic erythroblasts (see black arrows), while miR-486-3p mimic sample (ii) is enriched in cells from later steps of erythroid differentiation such as orthocromatic erythroblasts (see black arrows) together with nuclear extrusion phenomena (see asterisks) and enucleated reticulocytes/erythrocytes. 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<.05) (c–e) Flow cytometric detection (mean±S.E.M.; n=3) of CD36 and GPA at days 4 (c), 8 (d) and 12 (e) of erythroid unilineage culture post nucleofection with miR-486-3p/NegCTR inhibitors. *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<.05) (f, g) Flow cytometric data (mean±S.E.M.; n=3) for the expression of CD71, CD36 and GPA (early, intermediate and late erythroid markers, respectively) during erythroid unilineage culture. Data reported in the graphs display the percentages of CD71+CD36- (early erythroid) and CD71+CD36+ (intermediate erythroid cells) at day 4 (f) and the fractions of CD36+GPA- (intermediate), CD36+GPA+ (more mature) and CD36-GPA+ (late) erythroid cells (g) at day 8 of erythroid unilineage culture post nucleofection with MYBsiRNA/NegCTRsiRNA and/or miR-486-3p mimic/NegCTR mimic. *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: GPA, glycophorin A; MYBsiRNA, MYB-targeting siRNA; NegCTR mimic, negative control mimic; NegCTR inhibitor, negative control inhibitor; NegCTRsiRNA, negative control siRNA; n=number of experiments