Figure 3
Figure 3. Expression of miR-139 and miR-199a alters the balance of HSPC loss and expansion. (A) Murine 32D cells were infected with MSCV-BC vectors containing either, miR-139, miR-199a, miR-34a, miR-342, or no miRNA (EV) as control; 32D cells expressing miRNAs were expanded in IL-3 containing medium and the number of cells at indicated time points is plotted. (B) Equal numbers of 32D cells expressing miRNAs were mixed with control EV-expressing 32D cells and switched to CSF3-containing medium. Cell samples were taken at indicated time points and genomic DNA was isolated. The abundance of the different barcodes relative to the EV barcode signal and normalized to day 0 is depicted. Representative data of 3 independent experiments are shown. The error bars represent SD of 3 measurements. (C) 32D-CSF3R cells were switched from IL-3 to CSF3-containing medium on t = 0. Micrographs show the morphology of 32D-CSF3R cells on indicated time points of CSF3 treatment. Size bars indicate 10 µm. (D) CSF2-colony assays of 1 × 104 plated lin cells transduced with different miRNA expressing viruses. The number of CFU consisting of more than 50 cells after 7 days of growth relative to EV control is depicted. Data are from 2 independent experiments performed in triplicate and represent the mean and SD of 3 plates counted. (E) CSF3-colony assays were performed with 2 × 104 lin− cells transduced with miR-199a expressing viruses. The number of CFU consisting of more than 50 cells per colony is depicted. Data represent the mean and SD of 3 plates counted. (F) Same as (C). The number of CFU of 1 × 104 mouse Ercc1-/*292 and WT lin− cells transfected with LNA antagomiR against miR-139-3p and miR-199a-3p are shown. Data represent the mean and SD of 3 plates counted. (G) Lin− cells transduced with MSCV–miR-199a or MSCV-EV and mixed with untransduced WT lin− cells in a 1:10 ratio were transplanted in irradiated recipient mice (n = 8 per group). The change in lin−Kit+ (LK, progenitors) fraction in the BM 10 weeks posttransplantation and relative to the EV control is shown (*P < .05). The error bars represent SD of n = 8 mice. In panels (C-E), the significance was calculated with the Student t test (2-tailed, *P < .05).

Expression of miR-139 and miR-199a alters the balance of HSPC loss and expansion. (A) Murine 32D cells were infected with MSCV-BC vectors containing either, miR-139, miR-199a, miR-34a, miR-342, or no miRNA (EV) as control; 32D cells expressing miRNAs were expanded in IL-3 containing medium and the number of cells at indicated time points is plotted. (B) Equal numbers of 32D cells expressing miRNAs were mixed with control EV-expressing 32D cells and switched to CSF3-containing medium. Cell samples were taken at indicated time points and genomic DNA was isolated. The abundance of the different barcodes relative to the EV barcode signal and normalized to day 0 is depicted. Representative data of 3 independent experiments are shown. The error bars represent SD of 3 measurements. (C) 32D-CSF3R cells were switched from IL-3 to CSF3-containing medium on t = 0. Micrographs show the morphology of 32D-CSF3R cells on indicated time points of CSF3 treatment. Size bars indicate 10 µm. (D) CSF2-colony assays of 1 × 104 plated lin cells transduced with different miRNA expressing viruses. The number of CFU consisting of more than 50 cells after 7 days of growth relative to EV control is depicted. Data are from 2 independent experiments performed in triplicate and represent the mean and SD of 3 plates counted. (E) CSF3-colony assays were performed with 2 × 104 lin− cells transduced with miR-199a expressing viruses. The number of CFU consisting of more than 50 cells per colony is depicted. Data represent the mean and SD of 3 plates counted. (F) Same as (C). The number of CFU of 1 × 104 mouse Ercc1-/*292 and WT lin− cells transfected with LNA antagomiR against miR-139-3p and miR-199a-3p are shown. Data represent the mean and SD of 3 plates counted. (G) Lin− cells transduced with MSCV–miR-199a or MSCV-EV and mixed with untransduced WT lin− cells in a 1:10 ratio were transplanted in irradiated recipient mice (n = 8 per group). The change in lin−Kit+ (LK, progenitors) fraction in the BM 10 weeks posttransplantation and relative to the EV control is shown (*P < .05). The error bars represent SD of n = 8 mice. In panels (C-E), the significance was calculated with the Student t test (2-tailed, *P < .05).

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