Figure 2

MIR-99 overexpression inhibits chemotaxis of dHL-60 cells. (A) Construct for inducible miRNA expression. The miRNA and a dendra2 reporter (green) are controlled by TRE, Tetracyclin response element (red). The PGK promoter drives constitutive expression of puromycin resistance gene (cyan) and the rtTA, reverse tetracycline-controlled transactivator (red). (B) Quantification of MIR-99, MIR-233, and LET-7E in dHL-60 cell lines with/without induced expression of the vector control or MIR-99. The result from three independent experiments is normalized to U6 expression levels and presented as mean ± s.d., students’ t-test. (C) Cell populations gated for downstream cell profile analysis and transwell quantification. (D) Doxycycline-mediated induction in the vector or miR-99 expressing dHL-60 lines. Cells without doxycycline-mediated induction were used as a baseline. Percentage of cells with dendra2 levels above the baseline are shown. (E) Surface CD11b levels in vector or miR-99 expressing dHL-60 cells. Undifferentiated cells or differentiated cells stained with an isotype control were used as a baseline. Percentage of cells with CD11b levels above the baseline with or without doxycycline-mediated induction are shown. (F) Annexin V staining in the total cell population (excluding debris) of vector or miR-99 expressing dHL-60 cells. An unstained live sample is used to determine the baseline. Percentage of cells with AnnexinV levels above the baseline with or without doxycycline-mediated induction are shown. (C–F) One representative experiment of three independent trials is shown. (G–J) (G) Transwell migration of dHL-60 cells with/without induced expression of the vector control or MIR-99 toward fMLP, (H) phagocytosis of BioParticles by dHL60 cells represented as mean FITC, (I) H₂O₂ concentration in PMA-induced dHL60 cells, and (J) Quantification of dHL60 undergoing NETosis (left) and representative images (right). Scale bar, 100µm. Results are presented as mean ± s.d., from three independent experiments and normalized to vector, Kruskal–Wallis test or Mann-Whitney test.