![Figure 3. Functional analysis of miR-17/20/93/106 in primary mouse Lin− BM cells. (A) CFU-G assay of mouse lin− BM progenitors infected with MSCV–miR-17, -miR-20, -miR-93, miR-106 or empty vector control virus. Cells were plated in triplicate at densities of 1 × 104 cells per dish in 1 mL methylcellulose medium containing CSF3 (100 ng/mL). Colonies consisting of more than 50 cells were counted after 7 days of growth. Micrographs show size of CFU-CSF3 on day 7 after plating. Bar indicates 100 μm. Significance was calculated by comparing the samples with EV control and the different miRNAs with the Mann-Whitney test (asymptotic significance [2-tailed]). *P < .05. (B) Replating assay of mouse lin− BM progenitors infected with MSCV–miR-17, -miR-20, -miR-93, miR-106 or empty vector control virus. Cells were plated in triplicate at densities of 1 × 104 cells per dish in 1 mL methylcellulose medium containing IL-6 (10 ng/mL), IL-3 (supernatant 1/1000), SCF (10 ng/mL), CSF2 (10 ng/mL). Cells were isolated from dishes, counted and replated under the same conditions. Colonies from the second plating were counted after 7 days of growth. Micrographs show size of CFU's on day 7 after plating. Bar indicates 100 μm. Significance was calculated as described in panel A. (C) Mouse lineage negative progenitor cells isolated from the BM of C57BL/6 mice were infected with MSCV–miR-17, -miR-20, -miR-93, miR-106 or empty vector control virus. Recipient C57BL/6 mice were irradiated (8.5 Gy) and reconstituted with ∼ 20% GFP positive cells and ∼ 80% WT cells. Six weeks after transplantation, mice were killed and BM cells were isolated. Lin− cells were stained for flow cytometry analysis. The fold induction of the percentage GFP+ control (n = 4), miR-17 (n = 6), miR-20 (n = 4), miR-93 (n = 4), miR-106 (n = 6) Lin−; Sca-I+; c-Kit+/− cells in the BM compared with the input are shown. Significance was calculated by comparing the samples of mice transplanted with EV control and the different miRNAs with the Mann-Whitney test (asymptotic significance [2-tailed]). *P < .05 and **P = .057. (D) Ba/F3-CSF3R cells were infected with MSCV–miR-17, miR-20, miR-93, miR-106 or EV control viruses. Cells were factor deprived for 4 hours (t = 0) followed by CSF3 (100 ng/mL) stimulation for 10 minutes. Cells were washed 2 times with PBS and incubated in RPMI medium for 60 or 120 minutes. Samples for cell lysates were taken at indicated time points and analyzed by Western blotting using total and phospho-specific antibodies against ERK.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/118/4/10.1182_blood-2011-02-336487/4/zh89991174920003.jpeg?Expires=1724238034&Signature=FWB-n1ut831vkaRbKujUdmsMCxIexPAI~SgzvvWV7AAPiJ8rwYiBuR6Davt1mh34b3H4dyUCDe6TXmhlbBAXQOnlYzid3zr4nSsLYCa9-Lf6Vju7VUDaSKSnmdk2Avhyjq0l9iic7ZmYqwbNP9lC12B7KBpGsORV58C15oxhcZZq-NSmNXggY5~NPx0zbnPDth2eopkH6~4sDb3F3SvC0i~4OM5yJ0OpXM5rHpgzDNpgijnSRwBIKhvLCD-SNALK6iyDWWdwmYZNzOaGT1bJURu7g-OJ0edQb-b3QkC7LvOsHOQBadySB6gnTuNYLbkoSXjtEgbFTg8pKVWytTgIOQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Functional analysis of miR-17/20/93/106 in primary mouse Lin− BM cells. (A) CFU-G assay of mouse lin− BM progenitors infected with MSCV–miR-17, -miR-20, -miR-93, miR-106 or empty vector control virus. Cells were plated in triplicate at densities of 1 × 104 cells per dish in 1 mL methylcellulose medium containing CSF3 (100 ng/mL). Colonies consisting of more than 50 cells were counted after 7 days of growth. Micrographs show size of CFU-CSF3 on day 7 after plating. Bar indicates 100 μm. Significance was calculated by comparing the samples with EV control and the different miRNAs with the Mann-Whitney test (asymptotic significance [2-tailed]). *P < .05. (B) Replating assay of mouse lin− BM progenitors infected with MSCV–miR-17, -miR-20, -miR-93, miR-106 or empty vector control virus. Cells were plated in triplicate at densities of 1 × 104 cells per dish in 1 mL methylcellulose medium containing IL-6 (10 ng/mL), IL-3 (supernatant 1/1000), SCF (10 ng/mL), CSF2 (10 ng/mL). Cells were isolated from dishes, counted and replated under the same conditions. Colonies from the second plating were counted after 7 days of growth. Micrographs show size of CFU's on day 7 after plating. Bar indicates 100 μm. Significance was calculated as described in panel A. (C) Mouse lineage negative progenitor cells isolated from the BM of C57BL/6 mice were infected with MSCV–miR-17, -miR-20, -miR-93, miR-106 or empty vector control virus. Recipient C57BL/6 mice were irradiated (8.5 Gy) and reconstituted with ∼ 20% GFP positive cells and ∼ 80% WT cells. Six weeks after transplantation, mice were killed and BM cells were isolated. Lin− cells were stained for flow cytometry analysis. The fold induction of the percentage GFP+ control (n = 4), miR-17 (n = 6), miR-20 (n = 4), miR-93 (n = 4), miR-106 (n = 6) Lin−; Sca-I+; c-Kit+/− cells in the BM compared with the input are shown. Significance was calculated by comparing the samples of mice transplanted with EV control and the different miRNAs with the Mann-Whitney test (asymptotic significance [2-tailed]). *P < .05 and **P = .057. (D) Ba/F3-CSF3R cells were infected with MSCV–miR-17, miR-20, miR-93, miR-106 or EV control viruses. Cells were factor deprived for 4 hours (t = 0) followed by CSF3 (100 ng/mL) stimulation for 10 minutes. Cells were washed 2 times with PBS and incubated in RPMI medium for 60 or 120 minutes. Samples for cell lysates were taken at indicated time points and analyzed by Western blotting using total and phospho-specific antibodies against ERK.
