Journal: Immunity

Article Title: Autophagy-Dependent Generation of Free Fatty Acids Is Critical for Normal Neutrophil Differentiation

doi: 10.1016/j.immuni.2017.08.005

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Article Snippet: 32D-cl3 (32D) myeloblast cells, RRID CVCL_0119, are derived from Mus musculus , were purchased from ATCC and cultured in RPMI 1640 with 10% FCS, 2 mM L-glutamine,100 U/ml Pen-Strep, and 10 ng/ml IL-3 at 37°C, 5% CO 2 .

Techniques: Virus, Recombinant, Giemsa Stain, Labeling, Gene Expression, ATP Bioluminescent Assay, Cell Based Assay, Software

Journal: PLoS ONE

Article Title: Aciculatin Inhibits Granulocyte Colony-Stimulating Factor Production by Human Interleukin 1β-Stimulated Fibroblast-Like Synoviocytes

doi: 10.1371/journal.pone.0042389

Figure Lengend Snippet: (A, C, and D) FLS were incubated with 0, 1, 3, or 10 µM aciculatin (A1, A3, and A10) for 30 min, and then for 24 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. Whole cell extracts were then prepared for western blot analysis for the indicated proteins (A and C); equal amounts of cell culture media (“conditioned medium”) were collected and concentrated 10-fold (v/v) (lanes 1–4) or PBS only (lane 5), and then immunoprecipitated with 1 µg of anti-G-CSF antibody, followed by immunoblot analysis using anti-G-CSF antibody or anti-β-actin antibody (as an internal control) (D). (B) FLS were incubated with 0 or 10 µM aciculatin for 30 min, and then for 1 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. The DNA binding activity of the nuclear extracts was then examined in an electrophoretic mobility shift assay using a specific STAT3 DNA probe. (E) Ten-fold concentrated conditioned medium was prepared from FLS incubated with or without aciculatin, and then with IL-1β as in (D), or with IL-1β plus an anti-G-CSF antibody. 32Dcl3 cells were incubated for 10 days with a medium containing 50% of these different conditioned mediums. The cells were then were subjected to Wright-Giemsa staining to detect neutrophils (top row) or washed twice with PBS, incubated at 4°C for 45 min with anti-CD11b FITC-conjugated and anti-CD11a/CD18 PE-conjugated antibodies, and their fluorescence was analyzed by FACScan flow cytometry (bottom row). Magnification = ×100; scale bar = 20 µm. In (A) and (C), the extents of indicated proteins expression were quantitated using a densitometer with the Image-Pro plus software, and the relative levels were calculated as the ratios of proteins to GAPDH or β-actin protein levels. The results are expressed as the mean ± SEM, with n = 3. * p <0.05 and ** p <0.01 compared with the control group; # p <0.05 and ## p <0.01 for the comparisons of the groups indicated.

Article Snippet: Mouse myelomonocytic leukemia WEHI-3 cells and mouse bone marrow 32Dcl3 cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Incubation, Western Blot, Cell Culture, Immunoprecipitation, Control, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Staining, Fluorescence, Flow Cytometry, Expressing, Software