Journal: The Journal of Biological Chemistry

Article Title: Insulin Resistance Prevents AMPK-induced Tau Dephosphorylation through Akt-mediated Increase in AMPK Ser-485 Phosphorylation *

doi: 10.1074/jbc.M115.636852

Figure Lengend Snippet: Preventing AMPKSer-485 hyperphosphorylation by mutating Ser-485 restored AICAR-induced Tau dephosphorylation by insulin pretreatment. A, HK-532 cell were infected with a lentiviral vector expressing S485A AMPK mutants or control for 5 days. S485A mutant transfection reduced both basal and AICAR-induced Ser-485 phosphorylation by AICAR. The cells were treated without or with 50 nm insulin overnight and then treated with 1 mm AICAR for 2 h. B and C, cell lysates were examined for AMPK (B) and Tau (C) phosphorylation. D, HK-532 cells were transfected with S485A or S485D AMPK mutant, and the stable clones were selected by puromycin. AICAR-induced Tau dephosphorylation was examined after without or with 50 nm overnight insulin pretreatment. E, densitometry of the result of stable AMPK mutant transfection. *, p < 0.05; #, p < 0.005.

Article Snippet: Construction, Production, and Infection of Lentiviral Expression Vectors Human AMPKa1 cDNA was amplified from SH-SY5Y human neuroblastoma cell cDNA and directionally subcloned into the XhoI and NotI sites of the lentiviral expression vector pLVX-IRES-mCherry (Clontech).

Techniques: De-Phosphorylation Assay, Infection, Plasmid Preparation, Expressing, Mutagenesis, Transfection, Clone Assay

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: Molecular cell

Article Title: High-resolution in vivo identification of miRNA targets by Halo-Enhanced Ago2 Pulldown

doi: 10.1016/j.molcel.2020.05.009

Figure Lengend Snippet: A) Schematic of the Halo-Ago2 fusion protein covalently bound to a bead-conjugated HaloTag ligand. B) Ago2−/− immortalized MEFs transduced with MSCV-PIG, MSCV-PIG-Halo or MSCV-PIGHalo-Ago2 retroviruses were incubated with the HaloTag TMRDirect ligand and imaged. Notice the prevalently cytoplasmic localization of the Halo-Ago2 fusion protein. C) Ago2−/− MEFs transduced with retroviral vectors encoding HaloTag alone, full length Ago2 or the Halo-Ago2 fusion protein were transiently transfected with reporter plasmids expressing Firefly and Renilla luciferase and a plasmid expressing an shRNA against the Firefly luciferase. The ratio between Firefly and Renilla luciferase activity was measured 48 hours after transfection (upper panel). Whole-cell lysates from the same cells were probed with antibodies against Ago2 and β-actin (lower panel). Error bars: Mean ± SD. D) Schematic of the targeting strategy used to generate the Halo-Ago2 conditional knock-in allele. Halo: HaloTag; STOP: stop codon; IRES: internal ribosome entry site. E) Whole-cell lysates from mESCs with the indicated genotypes were probed with antibodies against Ago2, HaloTag and Tubulin. F) Outline of the strategy used to generate HEAP and input control libraries (upper panel) and a representative Halo-Ago2 binding site identified in mESCs (lower panel).

Article Snippet: Ago2 −/− MEFs were transduced with the MSCV-PIG ( Mayr and Bartel, 2009 ) (Addgene: 21654), MSCV-PIG-Halo, MSCV-PIG-Halo-Ago2 or MSCV-PIG-Ago2 retroviruses to generate cell lines stably expressing HaloTag, the Halo-Ago2 fusion or Ago2.

Techniques: Transduction, Incubation, Transfection, Expressing, Luciferase, Plasmid Preparation, shRNA, Activity Assay, Knock-In, Binding Assay

Journal: Molecular cell

Article Title: High-resolution in vivo identification of miRNA targets by Halo-Enhanced Ago2 Pulldown

doi: 10.1016/j.molcel.2020.05.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Ago2 −/− MEFs were transduced with the MSCV-PIG ( Mayr and Bartel, 2009 ) (Addgene: 21654), MSCV-PIG-Halo, MSCV-PIG-Halo-Ago2 or MSCV-PIG-Ago2 retroviruses to generate cell lines stably expressing HaloTag, the Halo-Ago2 fusion or Ago2.

Techniques: Reporter Assay, Recombinant, Western Blot, Blocking Assay, Knock-Out, Protease Inhibitor, SYBR Green Assay, Staining, Mass Spectrometry, Over Expression, Software