Journal: Cell reports

Article Title: Single-Cell RNA-Sequencing-Based CRISPRi Screening Resolves Molecular Drivers of Early Human Endoderm Development

doi: 10.1016/j.celrep.2019.03.076

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used in this study include: SOX17 antibody (1:300; R&D Systems, AF1924), OCT3/4 antibody (1:100; Santa Cruz Biotechnology, sc5279), FOXA2 antibody (1:300; Millipore EMD, 07–633), CDX2 antibody (1:300; BioGenex, MU392A-UC), HNF4A antibody (1:500; Abcam, ab41898), HHEX antibody (1:500; R&D Systems, MAB83771), TBX3 antibody (1:300; Santa Cruz Biotechnology, sc17871), PROX1 antibody (1:300; R&D Systems, AF2727), EPCAM antibody (1:1000; Biolegend, 324202).

Techniques: Recombinant, Staining, Transfection, DNA Library Preparation, Methylation, Software, Fluorescence, Microscopy

Journal: Journal of cell science

Article Title: Increased host ATP efflux and its conversion to extracellular adenosine is crucial for establishing Leishmania infection.

doi: 10.1242/jcs.239939

Figure Lengend Snippet: Fig. 4. Extracellular adenosine exerts effect via A2AR and A2BR. (A) Number of parasites per 100 cells was measured by DAPI staining in 24-h-infected PMφ in the presence of increasing concentrations of dipyridamole (5, 10 and 20 μM). (B) mRNA level expression of the adenosine receptors A1R, A2AR, A2BR and A3R was assessed by RT-PCR in uninfected and 8-h L. donovani-infected RAW cells. (C,D) RAW cells were infected with L. donovani promastigotes (L.d.) for the indicated time periods (0–24 h) and the expression of A2AR and A2BR were measured at the mRNA level by RT-PCR (C) and at protein level by immunoblotting (D). (E) RAW cells were infected with promastigotes for 8 h and then stained with anti-A2AR and anti-A2BR monoclonal antibodies followed by Texas Red- conjugated anti-A2AR secondary antibody and FITC-conjugated anti-A2BR secondary antibody. Nuclei were stained with DAPI and the cells were analysed under a confocal microscope. The intensity of staining for both antibodies were measured in each cell using ImageJ software and expressed as mean fluorescence intensity. (F) PMφ were pre-treated either with ZM241385 (1 μM) or MRS1754 (1 μM) or in combination for 30 min followed by infection with L. donovani for 24 h. The number of parasites per 100 macrophages were evaluated by DAPI staining. (G) Infected RAW 264.7 cells were pre-treated either with ZM241385 or MRS1754 and the expression of A2AR and A2BR were measured at protein level by immunoblotting. (H,I,J) Macrophages were treated with either LPS (100 ng/ml) or ZM241385 (1 μM) or MRS1754 (1 μM) or both ZM241385 and MRS1754 for 30 min followed by infection with L. donovani for 24 h. Culture supernatants were assayed for TNF-α and IL-12 (H) and IL-10 and TGF-β (I) and CCL-3 and CCL-5 (J) using ELISA. Bands were quantified by densitometry and are shown as bar graphs. Results are representative of three independent experiments and error bars are expressed as mean±s.d. n=3; ns, not significant, **P<0.01, ***P<0.001 (Student’s t-test).

Article Snippet: The antibodies against adenosine A2A receptor (Haschemi et al., 2007) (NBP139474, 7F6-G5-A2, 1:1000 for WB, 1:100 for microscopy; Lot# B-1) and Adenosine A2B receptor (Xu et al., 2018b) (NBP2-41312, 1:2000 for WB, 1:100 for microscopy; Lot# 811-1802) were purchased from Novus Biologicals (Centennial, Colorado, USA).

Techniques: Staining, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Bioprocessing, Microscopy, Software, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Journal of cell science

Article Title: Increased host ATP efflux and its conversion to extracellular adenosine is crucial for establishing Leishmania infection.

doi: 10.1242/jcs.239939

Figure Lengend Snippet: Fig. 6. L. donovani channels ATP of host macrophages for establishment of infection. L. donovani upon infecting host macrophage cell leads to upregulation of glycolysis and lactate production. ATP generated from the upregulated glycolysis, however, is exported out of the cell through pannexin-1 channel proteins. The increased extracellular ATP (eATP) thus produced gets degraded by the cell surface ATP-hydrolyzing ectonucleotidases CD39 and CD73, leading to generation of adenosine. A low level of adenosine deaminase (ADA), which deaminates adenosine (ADO), leads to sustained level of extracellular adenosine (eADO) resulting in an anti-inflammatory environment via signalling through the adenosine receptors A2AR and A2BR. A2AR signalling leads to downregulation of pro- inflammatory cytokines TNF-α and IL-12 whereas A2BR signalling leads to upregulation of anti-inflammatory cytokines IL-10 and TGF-β. Thus, Leishmania utilizes host cell ATP in favour of its own survival.

Article Snippet: The antibodies against adenosine A2A receptor (Haschemi et al., 2007) (NBP139474, 7F6-G5-A2, 1:1000 for WB, 1:100 for microscopy; Lot# B-1) and Adenosine A2B receptor (Xu et al., 2018b) (NBP2-41312, 1:2000 for WB, 1:100 for microscopy; Lot# 811-1802) were purchased from Novus Biologicals (Centennial, Colorado, USA).

Techniques: Infection, Generated, Produced