Journal: Cell discovery

Article Title: Insufficiency of DNA repair enzyme ATM promotes naive CD4 T-cell loss in chronic hepatitis C virus infection.

doi: 10.1038/s41421-018-0015-4

Figure Lengend Snippet: Fig. 3 DNA damage repair ATM signaling pathway in T cells of HCV-infected patients versus age-matched HS. a DNA damage sensor MRN complex expressions in CD4 T cells. Naive CD4 T cells were isolated from six HCV-infected patients and six age-matched HS, cultured in vitro without stimulation for 4 days, followed by real-time RT-PCR assay for MER11, RAD50, and NBS1 mRNA expression. b MRN protein expression detected by western blot in naive CD4 T cells derived from HCV patients vs. HS. Representative imaging and summary data from multiple subjects are shown. c ATM mRNA expressions in CD4 T cells. Naive CD4 T cells were isolated from the study subjects as indicated, cultured in vitro without stimulation for 4 days, followed by RT-PCR analysis for ATM mRNA level. d, e Flow cytometry and western blot analysis for ATM total and phosphorylated protein expression in naive CD4 T cells from HCV patients vs. HS. f–h ATM signaling molecule mRNA and protein expressions in CD4 T cells. Naive CD4 T cells were isolated from the study subjects, cultured in vitro without stimulation for 4 days, followed by RT-PCR analysis for P53, BRCA1, CHK1, and CHK2 mRNA level, western blot assay for their total and/or phosphorylated protein levels. pCHK2 expression was confirmed by flow cytometry analysis

Article Snippet: Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, which were blocked with 5% nonfat milk, 0.5% Tween-20 in Tris buffered saline, and incubated with the pATM (Ser1981) (D6H9), pBRCA1, pCHK1, and pCHK2 (Thr68) (C13C1) antibodies and βActin (8H10D10) antibodies (Cell Signaling, Danvers, MA).

Techniques: Infection, Isolation, Cell Culture, In Vitro, Quantitative RT-PCR, Expressing, Western Blot, Derivative Assay, Imaging, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry

Journal: Cell discovery

Article Title: Insufficiency of DNA repair enzyme ATM promotes naive CD4 T-cell loss in chronic hepatitis C virus infection.

doi: 10.1038/s41421-018-0015-4

Figure Lengend Snippet: Fig. 5 KU60019, an ATM inhibitor, induces DNA damage and cell apoptosis via inhibiting ATM pathway. a KU60019 inhibits ATM/pATM expressions in naive CD4 T cells. Representative imaging and summary data show western blot analysis for ATM/pATM expressions in naive CD4 T cells treated with 10 μM KU60019 or DMSO for 48 h. b KU60019 inhibits CHK2/pCHK2 expressions in naive CD4 T cells. c KU60019 enhances γH2AX expression in naive CD4 T cells. d KU60019 increases DNA damage foci (γH2AX/53BP1 co-localization) in naive CD4 T cells. e KU60019 increases T-cell apoptosis in naive CD4 T cells. Av, 7AAD, and active caspase-3 expressions are shown in naive CD4 T cells treated with KU60019 vs. DMSO control at 48 h

Article Snippet: Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, which were blocked with 5% nonfat milk, 0.5% Tween-20 in Tris buffered saline, and incubated with the pATM (Ser1981) (D6H9), pBRCA1, pCHK1, and pCHK2 (Thr68) (C13C1) antibodies and βActin (8H10D10) antibodies (Cell Signaling, Danvers, MA).

Techniques: Imaging, Western Blot, Expressing, Control

Journal: Journal of cellular physiology

Article Title: Adhesion G-protein coupled receptor 56 is required for 3T3-L1 adipogenesis.

doi: 10.1002/jcp.29079

Figure Lengend Snippet: FIGURE 8 Model illustrating adipogenesis in Gpr56 + ve and −ve cells. Model shows the cell membrane lipid bilayer and Gpr56 transmembrane protein ( + ve Gpr56) indicated by a black line passing through membrane that is absent in Gpr56 −ve cells (−ve Gpr56). Chemical treatment of preadipocytes allows differentiation in the presence of Gpr56. The link between Gpr56 and + ve stimulation of adipogenesis is unknown (?) but involves induction of Pparγ2 and C/ebpα (1) and other cryptic processes (2). Differentiation is indicated by morphological change from spindle like preadipocyte fibroblast cells to round adipocytes (3). In the absence of Gpr56 the change from fibroblast to adipocyte cells is blocked (4) and cells remain fibroblast like. Sustained levels of the known Pparγ2 and adipogenesis inhibitor, active β‐Catenin, occur (5). Reduced cell proliferation, adhesion and actin cytoskeleton and changes in extracellular matrix (ECM) gene expression are all proposed to contribute to inhibition of adipogenesis via β‐Catenin dependent (6) and independent (7) mechanisms [Color figure can be viewed atwileyonlinelibrary.com]

Article Snippet: Subsequently, membranes were washed in REVERTTM reversal solution and western blot analysis performed with antibodies to α‐GPR56 (Merck, H11), α‐PPARγ (C26H12), α‐C/EBP‐α (2295), α‐βACTIN (8H10D10), total β‐catenin (D10A8), active β‐catenin (D13A1; Cell Signaling Technologies®, Leiden, The Netherlands), or α‐AP‐2 (Santa Cruz Biotechnology Inc, C‐15) at 1:1,000 dilution.

Techniques: Membrane, Gene Expression, Inhibition