1. Volume 2, Issue 4, Pages 1048-1060 (October 2012)
An In Vivo Functional Screen Uncovers miR-150-Mediated Regulation of Hematopoietic Injury Response 
Brian D. Adams, Shangqin Guo, Haitao Bai, Yanwen Guo, Cynthia M. Megyola, Jijun Cheng, Kartoosh Heydari, Changchun Xiao, E. Premkumar Reddy, Jun Lu 
Cell Reports 
Volume 2, Issue 4, Pages (October 2012)
DOI: /j.celrep
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2. Cell Reports 2012 2, 1048-1060DOI: (10.1016/j.celrep.2012.09.014)
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3. Figure 1 Schematic of the In Vivo miRNA Screen
Donor bone marrow cells were transduced with a pooled miRNA expression library and transplanted into lethally irradiated recipient mice. Peripheral blood samples were collected at indicated weeks (Wk) posttransplantation. Mice were challenged with 5-FU 3 days after the week 11 collection and sampled at indicated days post-5-FU. Genomic DNA (gDNA) was isolated from peripheral blood and subjected to barcode analysis. See also Figure S1 and Tables S2 and S5.
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4. Figure 2 Barcode Analysis from the In Vivo Screen
(A) Barcodes were measured from red cell-lysed peripheral blood samples (effectively from all nucleated cells). The number of barcodes detected in each mouse at different time points is shown. Data were grouped by individual mice from a total of three independent cohorts of 15 mice.
(B) The number of unique barcodes detected in the screen cohort is shown for week 11 posttransplantation and day 10 post-5-FU. Data represent the number of barcodes that can be detected in at least the specified number of mice.
(C) Unsupervised clustering of barcode intensity primarily groups samples by recipient mice. A representative heatmap is shown in which log2-transformed data from samples collected at indicated weeks posttransplantation and days (D) post-5-FU were analyzed. Blue indicates lower signal, whereas red indicates higher signal.
See also Figure S2 and Table S5.
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5. Figure 3
In Vivo Screen Identifies Candidate miRNAs that Inhibit Hematopoietic Recovery
(A) A representative heatmap for consistency score evaluation of the screen. Comparisons between Day 10 and 22 post-5-FU time points to week 11 were calculated for each mouse (data in rows) and each miRNA barcode (data in columns). Not detected (N.D.) barcodes are shown in gray. Increased barcodes passing a change threshold are indicated in red, with those below the threshold in blue, and those not changed (neutral) in white.
(B) Barcode intensity decreases for miR-150 were compared in individual recipient mice between Day 10 or Day 22 post-5-FU and Week 11 pre-5-FU time points. A total of 13 mice had a detectable miR-150 barcode signal associated with these time points, with mouse identification (ID) shown at bottom. Day 22 samples for cohort 2 (mice 6–10) did not pass quality control and are not shown (see Experimental Procedures). Solid red line indicates no change. Dashed red lines indicate +0.6 and −0.6 of log2-fold changes that were used as the “change threshold” in (A). The circle (●) indicates that corresponding bar is not shown to the full height.
See also Figure S3 and Table S5.
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6. Figure 4
Forced Expression of miR-150 and Other Candidates Impairs Hematopoietic Recovery upon 5-FU Treatment
(A–C) Competitive recovery of transduced cells (GFP+) compared with nontransduced cells (GFP−) in mosaic transplantation hosts is shown for Mac1+ myeloid, B220+ B cell, CD3+ T cell lineages, and CD41+ platelets in the peripheral blood. Green arrow indicates the time point of maximal difference among the miR-150 (A), miR (B), miR-652 (C), and control cohorts in platelets, myeloid, B cell, and T cell lineages. GFP+/GFP− ratios were normalized to that of day −3 before 5-FU treatment. Error bars represent SD (n = 15 for A, n = 4 for B and C). ∗p < 0.05.
See also Figure S4.
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7. Figure 5
miR-150 Null Bone Marrow Confers Faster Peripheral Recovery upon 5-FU Injury
(A) The homeostatic levels of blood parameters are indistinguishable between wild-type (WT) and miR-150 knockout (KO) littermates. Complete blood counts are shown. WBC, white blood cells; HCT, hematocrit. Error bars indicate SD (n = 12).
(B) A schematic depicting competitive recovery assays comparing wild-type or miR-150 knockout cells with wild-type competitor bone marrow. Transplant recipients were tested for 5-FU injury response after donor bone marrow cells repopulated the recipient hematopoietic system and reached homeostasis. GFP was used to trace miR-150 knockout cells or those from wild-type littermates. CD45.1 and CD45.2-based tracing was used in a parallel experiment (see also Figure S5G). eGFP, enhanced green fluorescent protein.
(C) Competitive recovery of GFP+ miR-150 knockout cells or that of wild-type controls was compared with wild-type competitors (GFP−) and measured by peripheral GFP+/GFP− ratios. Data were from two independent cohorts and normalized to the ratios 3 days before 5-FU treatment. Error bars represent SD (n = 12). Green arrow indicates the time point with maximal difference between WT and KO cohorts for myeloid cells. ∗p < 0.05.
See also Figure S5.
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8. Figure 6
Forced Expression of miR-150 Inhibits Hematopoietic Progenitor Activity upon 5-FU-Induced or Irradiation Injury
(A) Bone marrow cells transduced with miR-150 or a control vector (GFP+), and those not transduced (GFP−), were sorted from transplant recipients and were subjected to colony formation assays. Both mice without 5-FU treatment and 6 days post-5-FU were analyzed. Results were from three pairs of miR-150 and control recipients. ∗p < 0.05.
(B) Bone marrow clonogenic potential from wild-type and miR-150 knockout mice was similarly assayed with or without 5-FU treatment (n = 4). ∗p < 0.05.
(C) Spleen colony formation potential in miR-150 or control transplant recipients. Wild-type bone marrow was transduced with miR-150 or control vector. GFP+ cells were sorted, and 1.5 × 104 cells were injected into irradiated recipients. Ten days post transplantation, the number of spleen nodules was counted, and spleen weight was also determined. Images are representative of spleen morphology for each cohort (n = 4). ∗p < All figure error bars depict the SD.
(D) Lin−Kit+Sca+ HSPCs were transduced with miR-150 or a control vector, treated in vitro with or without 5-FU, and assayed at indicated days. AnnexinV+ cells were analyzed after gating on 7AAD− population for both transduced (GFP+) and untransduced (GFP−) cells in the same culture. Error bars represent SD (n = 3). ∗p < 0.05.
(E) Bone marrow cells from miR-150 or control vector recipients were analyzed for apoptosis by examining the AnnexinV+ 7AAD− population in both Lin− and Lin+ populations, 6 days after 5-FU treatment in vivo. Error bars represent SD (n = 3).
See also Figure S6 and Table S4.
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9. Figure 7
Heterozygous Loss of c-myb Partially Phenocopies miR-150-Forced Expression
(A) Schematic of competitive recovery of c-myb heterozygous knockout cells. CD45.1/.2+ MybF/+Mx1Cre+ bone marrow cells or MybF/+Mx1Cre− cells were cotransplanted with CD45.2+ wild-type cells. After mice recovered from pIpC-induced deletion of c-myb, mice were challenged with 5-FU, and competitive recovery was measured in peripheral blood.
(B) The ratios of the test cells (CD45.1/.2) to competitor cells (CD45.2) were measured in peripheral blood before and after 5-FU treatment. The 45.1/45.2 ratios were normalized to the day −3 time point. Error bars represent SD (n = 4). ∗p < 0.05.
See also Figure S7.
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10. Figure S1
miRNA Library, Screen Platform, and Expression Changes Associated with 5-FU Treatment, Related to Figure 1
(A) Unsupervised clustering of miRNA profiling data from bone marrow samples of mice treated with 5-FU versus untreated controls. The heatmap depicts 168 miRNAs (rows) that passed the detection threshold cutoff, from five untreated control samples (4-8) and three 5-FU treated samples (1-3). Samples 1A and 1B are technical replicates. Further analysis indicated that 92 miRNAs were differentially expressed between the two cohorts with a false discovery rate of < 5%.
(B) The miRNA expression library was cloned by PCR amplifying genomic DNA (gDNA) from regions containing miRNA hairpin(s) and flanking sequences. The cloning was performed by a two-step strategy with the non-ligase-based Gateway cloning. PCR fragments were first cloned into a Gateway entry vector, and the inserts were then recombined into a pMSCV-based destination vector. To make the pooled library, DNA was prepared from a mixture of singly-inoculated clones for each miRNA. Retroviral supernatant then made from this mixed DNA pool.
(C) Detection of barcode signals were performed using a bead-based system. Construct inserts were PCR-amplified from genomic DNA of cells transduced with the library. One of the PCR primers was labeled with biotin, and thus could be bound by streptavidin-phycoerythrin (SA-PE). Beads of unique color identifiers were coupled with antisense oligos to detect barcode sequences, which could be deconvoluted and quantified based on flow-cytometry.
(C and D) Specific and reproducible detection of barcode signals are depicted. (C) HL-60 cells were transduced with a control vector (no barcode), miR-29a alone, or with the miRNA expression library. Genomic DNA of transduced cells was analyzed for barcode abundance. Before hybridization to detector beads, PCR products were spiked with specific hybridization controls to control for efficiency of detection. (D) The same genomic DNA was analyzed by two independent PCR reactions. Data shown are log2 values of raw signals. Each dot represents signal from one barcode detector.
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11. Figure S2
Blood Parameter and Engraftment Analysis of miRNA Library Recipients, Related to Figure 2
(A and B) Peripheral blood parameters largely stabilized in the screen cohort before the treatment of 5-FU. (A) Complete blood counts from the 15 screen cohort recipients. Error bars represent standard deviation. (B) GFP+ cell percentages (representing transduced cells) were shown for a representative cohort. Each line represents a single mouse. CD41+ platelets, Mac1+ myeloid cell, B220+ B cell and CD3+ T cells were measured.
(C) Complete blood counts of the screen cohort during 5-FU-induced injury response are shown. Days pre or post-5FU are shown on the horizontal-axis. Day −3 was the week 11 time point post-transplantation. Error bars represent standard deviation.
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12. Figure S3
Barcode Analysis in miR Library Recipients, Related to Figure 3
(A–C) Histograms of log2 barcode intensities are shown for (A) screen samples detected with barcode detectors for the miRNA library, or (B) water samples (using water as PCR template) detected with barcode detectors for the miRNA library, or (C) screen samples detected with unrelated detectors. A representative screen cohort is shown. Note the bimodal distribution with the left peak representing detection background and the right flat peak representing signals.
(D) The numbers of unique barcodes detected in the complete screen cohort 3 weeks post-transplantation are shown. Data represent the number of barcodes that can be detected in at least the specified number of mice.
(E) The same but larger heatmap, as shown in Figure 2E, is depicted. Each row represents a miRNA, and each column represents a mouse at a given time point. See Figure 2E legend for more details.
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13. Figure S4
Analysis of Blood Parameters in Mice with Forced miR-150 Expression before 5FU Treatment, Related to Figure 4
(A) Complete blood counts from mice expressing miR-150 or the vector control indicate peripheral blood parameters largely stabilized before the treatment of 5-FU (N = 5 per group). Error bars represents standard deviation. A representative cohort is shown.
(B) GFP+/GFP− ratio of platelets, myeloid cells, B- and T cells from a representative cohort is shown. Data were normalized to the ratio at week 3 post transplant. Error bars represent standard deviation.
(C) Raw GFP+/GFP− ratios within each peripheral blood compartment at wk 3 (left panel) and wk 11 (right panel) post transplantation in both miR-150 (blue bars) and vector control (red bars) mice. Error bars represent standard deviation of n = 10.
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14. Figure S5
Baseline Hematopoietic Parameters of miR-150 Knockout Mice and Analysis of Competitive Transplantation Assays, Related to Figure 5
(A) Assessment of CD19+ B220+ CD5+ peritoneal B cell populations in wild-type (WT) and knockout (KO) mice. A representative mouse pair is shown.
(B) Peripheral Mac-1, B220, and CD3 profile of miR-150 KO and WT littermates. A representative mouse pair is shown.
(C) Panel of B (B220+), T (CD3+), myeloid (Mac-1+), and erythroid (Terr119+) populations in the bone marrow and spleen of miR-150 WT and KO mice. Compartment percentages are denoted as % of total organ site. Data from a representative mouse pair are shown.
(D) Percent CD41+ CD61+ c-kit− megakaryocytes from the spleens of miR-150 +/+, +/−, and −/− mice (n = 3). Error bars represent standard deviation. N.S = not significant compared to WT cohort.
(E) Peripheral blood analysis of the miR-150-GFP knockout competition assay. The peripheral GFP+/GFP- ratio largely stabilized before 5-FU analysis. Ratios shown were normalized to those at 3 weeks post-transplantation. Error bars indicate standard deviation. N = 12.
(F and G) Peripheral blood analysis of miR-150- knockout competition assay using CD45.1 and CD45.2 markers. The effect of miR-150 loss on hematopoietic recovery was measured by competitive recovery using the CD45.1/CD45.2 tracing scheme. (F) The peripheral CD45.1/CD45.2 ratio largely stabilized before 5-FU analysis. Ratios were normalized to that of weeks 3 post-transplantation. (G) The competitive recovery of both miR-150 homozygous knockout and wild-type cohorts are shown. The CD45.1/CD45.2 ratios were normalized to the time point before 5-FU treatment (day −3). Error bars represent standard deviation. N = 9.
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15. Figure S6
Analysis of Apoptosis and Endogenous miR-150 Levels in Hematopoietic Cells after 5-FU-Induced Injury, Related to Figure 6
(A) HSPCs (Lin− Kit+ Sca+) were transduced with miR-150 or a control construct, cultured in vitro with or without 5-FU (250nM), and analyzed after the indicated days post 5-FU addition. Representative flow cytometry plots (for data in Figure 6D) assessing GFP (labeling transduced cells) and AnnexinV populations are shown.
(B) Representative flow cytometry plots are shown for data in Figure 6E. Bone marrow cells from miR-150 or control-vector recipients (6 days after 5-FU treatment) were analyzed for apoptosis by examining the AnnexinV+ population, after gating on 7AAD− events, and further gating on either Lin− or Lin+ populations. The right panel depicts control staining conditions where bone marrow samples from control mice were unstained, or stained with lineage cocktail (Lin) or AnnexinV. AnnexinV+ percentages are shown in the Lin− (middle column) and Lin+ (right column) populations, respectively. The left panel depicts the same staining and gating parameters for miR-150 and miR-Control transplant recipients.
(C) miR-150 and c-myb RNA levels were measured by quantitative RT-PCR in potential progenitor populations of untreated wild-type mice or mice treated with 5-FU for 6 days. Specifically, c-Kit+, c-Kit− Lin− Sca-1−, and c-Kit− Lin− Sca-1+ populations were sorted from whole bone marrow, and assessed for miR-150 and c-myb levels, with data normalized to U6 and 18S levels respectively. Data shown reflect two mice per treatment condition, with error bars depicting standard deviations from technical replicates.
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16. Figure S7
Detailed Analysis of Heterozygous Loss of c-myb, Related to Figure 7
(A) Left: genotyping of donor mice, showing Mybfl/+Cre+ and Mybfl/+Cre− genotypes. Right: deletion of a single copy of c-myb was verified in transplantation recipients before 5-FU treatment. Deleted (D), floxed (F) or wild-type (+) bands were amplified in the same genotyping PCR reaction. Note that only Mybfl/+Cre+ recipients contained a deleted band, with subsequent loss of the majority of the floxed allele signal.
(B) The peripheral CD45.1 / 45.2 ratio normalized to that of weeks 3 post-transplantation, before 5-FU treatment. Error bars represent standard deviation.
(C) Complete blood counts from each mouse cohort show similar and expected responses before 5-FU treatment. Error bars represent standard deviation.
(D) Bone marrow cells from pIpC treated Mybfl/+Cre+ and Mybfl/+Cre- mice were subjected to myeloid colony formation assays. Both mice without 5-FU treatment and 6 days post-5-FU were analyzed, with 2 mice in each group. Each bar represents an individual mouse.
(E) Competitive recovery of GFP+ Mybfl/+Mx1Cre+ bone marrow cells or Mybfl/+Mx1Cre− cells co-transplanted with GFP− wild-type cells. After mice recovered from pIpC-induced deletion of c-myb (3 weeks), mice were challenged with 5-FU and competitive recovery of platelets was measured. Resulting platelet GFP+/GFP− ratios were normalized to the Day −3 time-point. Error bars represent standard deviation of (N = 5). ∗p < 0.05.
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