Fig. 3

miR-183-5p|+2 is functionally different from the other two isoforms. a-c Effect of 5’isomiR-183-5p overexpression on cell viability. a MDA-MB-231, b HCC-1806 and c BT-549 cells were seeded into clear-bottom 96-well black plates and transfected with miRNA mimics or siAllstar. 72h after transfection, cells were stained with Hoechst 33342 and propidium iodide and quantified using Molecular Devices Microscope IXM XLS. The number of viable cells was measured by counting cell nuclei positive for Hoechst and negative for propidium iodide staining with Molecular Devices Software. Values were normalized to the respective control. Data are presented as mean ± SD, n = 3 (each derived from the median of 6 technical replicates). *p < 0.05, **p < 0.01, ***p < 0.001 compared to control, one sample t-test. d-f Effect of 5’isomiR-183-5p overexpression on cell cycle. d MDA-MB-231, e HCC-1806 and f BT-549 Cells were transfected with miRNA mimics or siAllstar for 48 h. After transfection, the cells were starved with 0% FBS medium for 24 h to partially synchronize the distribution of cell cycle, and subsequently were released with full growth medium for 24 h. Afterwards, the cells were pulsed with BrdU for 2 h to label cells actively synthesizing DNA during that time period. FITC conjugated anti-BrdU antibody was used to stain the cells with BrdU incorporation. 7-AAD, a dye binding to total DNA, was coupled with BrdU staining. With this combination, two-color flow cytometry analysis was applied to determine cell cycle distribution (G0-G1, S, and G2-M phase). Data are presented as mean ± SD, n = 3 (each derived from the median of 3 technical replicates). Statistical analysis: ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001 compared to control