Journal: Cell stem cell

Article Title: Gli1 + mesenchymal stromal cells are a key driver of bone marrow fibrosis and an important cellular therapeutic target

doi: 10.1016/j.stem.2017.03.008

Figure Lengend Snippet: (A) Bigenic Gli1CreERt2;tdTomato mice received tamoxifen (3x10mg p.o.) and were lethally irradiated 10 days after the last tamoxifen dose and transplanted with 2x105 c-kit purified bone marrow (BM) cells from wild type littermates that had been transduced with either control (n=10) or Jak2(V617F) (n=10) cDNA (both MCSV-IRES-GFP retroviral backbone vector). Mice were injected with GANT61 (50mg/kg body weight) or vehicle (corn oil / ethanol) every other day between 8 and 17 weeks after transplantation (control + vehicle n=3, 2 males; control+GANT61 n=5, 3 males; Jak2(V617F) + vehicle n=4, 3 males; Jak2(V617F) + GANT61 n=4, 3 males). (B) Grading of reticulin fibrosis. ***p<0.001 versus all other groups by one way ANOVA with posthoc Tukey; mean±SEM. (C) Spleen weight was determined as a percentage of the body weight. *p<0.05, **p<0.001 by one way ANOVA with posthoc Tukey; mean±SEM. (D) Representative images of sternal bones. Scale bars 50μm, inserts 25μm. (E) Quantification of all alpha smooth muscle actin expressing cells (α-SMA+), Gli1 cells (tdTomato+) and Gli1-derived myofibroblasts (α-SMA+/tdTomato+) in the BM of mice transplanted with Jak2(V617F) (Jak2) or control that have been treated with GANT61 or vehicle. ***p<0.001 by one way ANOVA with posthoc Tukey; mean±SEM. (F) Representative flow cytometric plots and quantification of Gli1+ cells and within the lineage depleted BM. *p<0.05 by one way ANOVA with posthoc Tukey; mean±SEM. (G) Analysis of long-term (LT; linlowSca1+ckit+CD48-CD150+) and multipotent progenitor cells (MPP, linlowSca1+ckit+CD48+CD150-) as well as erythroid cells (Gr1-CD11b-CD3-CD19-Ter119+) by flow cytometry. *p<0.05, **p<0.01 by one way ANOVA with posthoc Tukey; mean±SEM. (H) c-kit+ purified HSPCs from wild type mice were transduced with either control or Jak2(V617F) cDNA (both MCSV-IRES-GFP retroviral backbone vector). 48 hours after transduction, cells were treated with GANT61 or vehicle. The gene marking (GFP+) was quantified by flow cytometry 48 hours after treatment. ***p<0.001 versus all other groups by one way ANOVA with posthoc Tukey; mean±SEM. (I) Quantification of the mean fluorescence intensity (MFI) and representative flow cytometric analysis of levels of phospho-STAT5 (p-STAT5) in c-kit+ cells transduced with Jak2(V617F) or control (GFP+) 48 hours after treatment with GANT61 or vehicle. GFP+ cells are shown in the histogram. ***p<0.001 versus all other groups by one way ANOVA with posthoc Tukey; mean±SEM. (J) c-kit+ purified HSPCs were transduced with Jak2(V617F) or control cDNA, were sort-purified by GFP-expression 48 hours after transduction and treated with GANT61 or vehicle 24 hours later. 24 hours after treatment, cells were harvested for RNA isolation and qt-RT-PCRs. Relative mRNA expression for Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), Akt and Gli1 is shown. *p<0.05 ns=non-signifcant by one way ANOVA with posthoc Tukey; mean±SEM. See also Figure S4.

Article Snippet: The murine Jak2 cDNA was cloned into the retroviral vector MSCV-IRES EGFP (a gift from Tannishtha Reya Addgene #20672).

Techniques: Irradiation, Purification, Transduction, Control, Retroviral, Plasmid Preparation, Injection, Transplantation Assay, Expressing, Derivative Assay, Flow Cytometry, Fluorescence, Isolation

Journal: Cell stem cell

Article Title: Gli1 + mesenchymal stromal cells are a key driver of bone marrow fibrosis and an important cellular therapeutic target

doi: 10.1016/j.stem.2017.03.008

Figure Lengend Snippet: Bigenic Gli1CreER;tdTomato mice were injected with tamoxifen (3x10mg p.o.) at 8 weeks of age, lethally irradiated at 10 days after the last tamoxifen dose and received c-kit enriched hematopoietic stem cells from wildtype littermates expressing either Thrombopoietin-cDNA (ThPO, n=5, 3 males) or control-cDNA (control, n=4, 3 males; both lentiviral SFFV-iGFP vector backbone). Mice were sacrificed at 70 days after transplantation. Gli1+ cells were sort-purified as lin-GFP-dtTomato+ and subjected to RNA sequencing. (A) Principal component analysis (PCA). (B) Heatmap representation with hierarchical clustering (C) Significantly directional enriched (median FDR >0.05) pathways. (D) Fragments per kilobase of exon per million fragments mapped (FPKM) values of CXCL4 in control and ThPO group; mean±SEM. (E) Cytokine analysis in serum and cytokine-free medium supernatant of ckit+ cells expressing ThPO or control (lentiviral SFFV-iGFP vector backbone) or Jak2(V617F) or control (both MCSV-IRES-GFP retroviral backbone). n=2. (F) FPKM value of stromal derived factor 1 (SDF 1/ CXCL12) in control and ThPO group; mean±SEM. (G) Representative images of bone marrow from Gli1CreERt2;tdTomato mice transplanted with bone marrow from wildtype littermates expressing either ThPO or control-cDNA stained with the neutral lipid staining LipidTOX. Scale bars 50μm. Arrow indicating LipidTOX positive Gli1+ cells. (H) Model for stroma-hematopoiesis interactions in bone marrow fibrosis. See also Figure S5.

Article Snippet: The murine Jak2 cDNA was cloned into the retroviral vector MSCV-IRES EGFP (a gift from Tannishtha Reya Addgene #20672).

Techniques: Injection, Irradiation, Expressing, Control, Plasmid Preparation, Transplantation Assay, Purification, RNA Sequencing, Retroviral, Derivative Assay, Staining

Journal: Hepatology (Baltimore, Md.)

Article Title: Genome-wide microRNA and messenger RNA profiling in rodent liver development implicates mir302b and mir20a in repressing transforming growth factor-beta signaling.

doi: 10.1002/hep.26252

Figure Lengend Snippet: Fig. 2. The dynamic expression of miRNA genes during liver develop- ment. (A) Based on the expression pattern of each miRNA, miRNA clus- ters consisting of expression enriched in foregut (Cluster A), hepatoblast (Cluster H), and adult liver (Cluster L) were identified. (B) Validation of mir302b and mir24 expression by qRT-PCR. U6 was used as an endoge- nous control. All data presented are representative of at least three inde- pendent experiments.

Article Snippet: The mir302b overexpression vector, pCMV-mir302b-IRES-GFP (302b_OE), and its control vector, pCMV-mir-IRESGFP (Ctrl_OE), were purchased from Origene.

Techniques: Expressing, Biomarker Discovery, Quantitative RT-PCR, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Genome-wide microRNA and messenger RNA profiling in rodent liver development implicates mir302b and mir20a in repressing transforming growth factor-beta signaling.

doi: 10.1002/hep.26252

Figure Lengend Snippet: Fig. 3. mir302b is expressed in the endoderm at the time of organogenesis. (A,C) mir302b expression in early streak embryo (E6.5); lateral view (A) and transverse section (C). (B,D) mir302b expression in E7.5 embryo; lateral view (B) and transverse section (D). (E,F) Lateral view of mir302b expression in 3-somite stage embryo (E8.5) (E) and transverse sections (F). a, anterior; p, posterior; Epi, epiblast; m, mesoderm; ve, visceral endoderm; en, endoderm; PS, primitive streak; n ect, neural ectoderm; ect, ectoderm; Fg, foregut. All data presented are representative of at least three independent experiments.

Article Snippet: The mir302b overexpression vector, pCMV-mir302b-IRES-GFP (302b_OE), and its control vector, pCMV-mir-IRESGFP (Ctrl_OE), were purchased from Origene.

Techniques: Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Genome-wide microRNA and messenger RNA profiling in rodent liver development implicates mir302b and mir20a in repressing transforming growth factor-beta signaling.

doi: 10.1002/hep.26252

Figure Lengend Snippet: Fig. 4. mir302b is expressed in the whole endoderm at E8.75. (A-D) mir302b expression in E8.75 embryo; lateral view (A) and transverse sections (B-D). nt, neural tube; ht, heart; a, allantois. All data presented are representative of at least three independent experiments.

Article Snippet: The mir302b overexpression vector, pCMV-mir302b-IRES-GFP (302b_OE), and its control vector, pCMV-mir-IRESGFP (Ctrl_OE), were purchased from Origene.

Techniques: Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Genome-wide microRNA and messenger RNA profiling in rodent liver development implicates mir302b and mir20a in repressing transforming growth factor-beta signaling.

doi: 10.1002/hep.26252

Figure Lengend Snippet: Fig. 5. mir302b and mir20a tar- get genes contributing to Tgfb sig- naling. (A) 30UTR sequences from Tgfbr2 and Kat2b showing the pre- dicted miRNA binding regions were cloned into luciferase vectors. The numbers indicate the distance (base) to stop codon of each gene. Mutated versions are shown below. (B) Relative luciferase activity of Tgfbr2 or Kat2b wildtype (WT), mu- tant (Mut) and backbone (Vector) reporters transfected into HEK293T cells containing mir302b overex- pression vector (302b_OE, :) or its control vector (Ctrl_OE, ), or mir20a knockdown vector (20a_KD, ;) or its control vector (Ctrl_KD, þ). (C) Western blot showing levels of Tgfbr2 in HEK293T cells transfected with 302b_OE, Ctrl_OE, 20a_KD, or Ctrl_KD. Numbers under each band show relative quantification. All data presented are representative of at least three independent experiments.

Article Snippet: The mir302b overexpression vector, pCMV-mir302b-IRES-GFP (302b_OE), and its control vector, pCMV-mir-IRESGFP (Ctrl_OE), were purchased from Origene.

Techniques: Binding Assay, Clone Assay, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Control, Knockdown, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Genome-wide microRNA and messenger RNA profiling in rodent liver development implicates mir302b and mir20a in repressing transforming growth factor-beta signaling.

doi: 10.1002/hep.26252

Figure Lengend Snippet: Fig. 6. mir302b and mir20a repress TGFb signal transduction. Luciferase activity of the TGFb responsive reporter, 3TP-lux, 48h after transfec- tion of HEK293T cells with mir302b_OE (:), Ctrl_OE (), mir20a_KD (;), Ctrl_KD (þ), pBOS (), pBOS-Tgfbr2 (þ), or pBOS-Tgfbr2(DN) (þ) in the presence (þ) or absence () of TGFb. Mean values in row with same letter are not significantly different. Values in row with different letters are significantly different.

Article Snippet: The mir302b overexpression vector, pCMV-mir302b-IRES-GFP (302b_OE), and its control vector, pCMV-mir-IRESGFP (Ctrl_OE), were purchased from Origene.

Techniques: Transduction, Luciferase, Activity Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Genome-wide microRNA and messenger RNA profiling in rodent liver development implicates mir302b and mir20a in repressing transforming growth factor-beta signaling.

doi: 10.1002/hep.26252

Figure Lengend Snippet: Fig. 7. Ectopic expression of mir302b in ESC differentiation. A lentiviral vector-based system was employed to ectopically express mir302b. Lentivirus generated from the control vector (pCDH) or mir302b overexpression vector (pCDH302b) was added at day 6 (d6) of differentiation. The expression of mir302b, mir20a, and Tgfbr2 during ESC differentiation with pCDH or pCDHmir302b virus was analyzed by qRT-PCR. All data presented are representative of at least three independent experiments.

Article Snippet: The mir302b overexpression vector, pCMV-mir302b-IRES-GFP (302b_OE), and its control vector, pCMV-mir-IRESGFP (Ctrl_OE), were purchased from Origene.

Techniques: Expressing, Plasmid Preparation, Generated, Control, Over Expression, Virus, Quantitative RT-PCR

Journal: Hepatology (Baltimore, Md.)

Article Title: Genome-wide microRNA and messenger RNA profiling in rodent liver development implicates mir302b and mir20a in repressing transforming growth factor-beta signaling.

doi: 10.1002/hep.26252

Figure Lengend Snippet: Fig. 8. Forced expression of mir302b represses liver development in ESC differentiation. The expression of liver markers, Alb, Afp, Hnf4a, Ttr, Hpx, and Serpina1a, were detected at the end of the differentiation. All data presented are representative of at least three independent experiments.

Article Snippet: The mir302b overexpression vector, pCMV-mir302b-IRES-GFP (302b_OE), and its control vector, pCMV-mir-IRESGFP (Ctrl_OE), were purchased from Origene.

Techniques: Expressing