Journal: Annals of the Rheumatic Diseases

Article Title: MxA as a clinically applicable biomarker for identifying systemic interferon type I in primary Sjögren's syndrome

doi: 10.1136/annrheumdis-2012-202552

Figure Lengend Snippet: Flow cytometric analysis of CD14 monocytes from healthy controls (HCs) and primary Sjögren's syndrome (pSS) patients. Correlation plots shown between interferon (IFN) scores for all study subjects and (A) CD64, (B) CD169 and (C) B cell-activating factor (BAFF) protein levels, respectively. Relative mean fluorescent intensity (MFI) for (D) CD64 (E) CD169 and (F) BAFF protein levels are shown for CD14 monocytes from HC (n=27) and pSS patients, stratified in IFN type I signature negative (IFNneg) (n=14) and IFN type Isignature positive (IFNpos) (n=21) patients. Each symbol represents an individual sample; horizontal lines represent the median. Relative expression was calculated as protein-specific staining (MFI)/isotype control (MFI). The correlation coefficients (r) and p values are shown. For correlations on CD64 and CD169, Spearman's rho correlation test was used and to compare medians, the Mann–Whitney U test was used. For BAFF, Pearson's correlation test was used and to compare means, the independent t test was used.

Article Snippet: Membrane staining was performed with fluorescently labelled antibodies: anti-CD14 (APC/Cy7; BD Biosciences, California, USA), and either anti-CD64 (PE; Serotech, Toronto, Canada) or CD169 (Alexa Fluor-647; Serotech) were incubated in the dark for 20 min.

Techniques: Expressing, Staining, Control, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: Exosomes secreted by ST3GAL5 high cancer cells promote peritoneal dissemination by establishing a premetastatic microenvironment

doi: 10.1002/1878-0261.13524

Figure Lengend Snippet: ST3G5 high ‐cExo augments CD169 expression in MΦs, which accelerates exosome uptake. (A–C) Cell surface proteins of wt or ST3G5 KO 44As3 cells (A, B) or B16 cells (C) were labeled by membrane‐impermeable biotin, and exosomes were collected, added to MΦs (cExo; 5 μg·mL −1 ), and incubated for 12 h. The MΦs were fixed and immunofluorescence stained by streptavidin and phalloidin (A, representative images of five fields in each), or subjected to FACS analysis of biotin (B). (C) Uptake of biotin‐labeled B16‐cExo in MΦs was assessed by immunostaining. Biotin incorporated in MΦs was expressed as the percentage of red fluorescence area in each cell. Random 100 cells were analyzed. (D) Biotin‐labeled wt (a) or ST3G5 KO (b) 44As3 cells were mixed with MΦs and co‐cultured for 24 h. Cells were fixed, and immunofluorescence stained by anti‐CD11b antibody (green) and streptavidin (red). Representative images of the co‐culture with 44As3 were shown. Arrows indicate biotin incorporated MΦs. Asterisks indicated biotin‐labeled 44As3 cells. Bar, 100 μm. (c) Co‐cultured cells were collected, and CD11b + MΦs were sorted by FACS, and subjected to immunoblot analysis of biotin amount. Larger amount of biotin + proteins of wt 44As3 cell was transferred into MΦs (left panel). Equal amount of biotin‐labeled proteins was observed in wt and ST3G5 KO ‐44As3 cells (right panel). (E, F) MΦs were incubated with biotin‐labeled 44As3‐cExo (E) or B16‐cExo (F) (5 μg·mL −1 ), and CD169 expression in biotin‐uptake MΦs was analyzed. (G, H) Biotin + B16‐cExos were added to immature DCs prepared from bone marrow for 2 days (5 μg·mL −1 ), and biotin‐uptake and CD169 expression were analyzed as above. (I) MΦs or iDC were incubated with biotin + exosomes of wt‐44As3 or ‐B16 cells (5 μg·mL −1 ) with/without soluble GM3 (20 μ m ). Incorporated biotin was evaluated by FACS. (J) C57BL/6 mice were i.p injected by wt or ST3G5 KO B16‐cExo labeled with ExoSparkler (red, 30 μg). As the control, exosomes were not injected (Exo(−)). Representative appearance of the mesentery and omentum. Arrows indicate the cExo in MS. Bar, 1 mm (mesentery), 2.5 mm (omentum). Right: MΦs were gated by CD11b + , and ExoSparkler was evaluated. Five mice were examined in each group. B, E–I; Results of three independent experiments. B, C, E–J; mean ± SD. * P < 0.01, Student's t ‐test.

Article Snippet: Purchased antibodies were as follows: α‐tubulin (Sigma‐Aldrich, St. Louis, MO, USA, RRID:AB_477593), HIF1α (GeneTex, Irvine, CA, USA, RRID:AB_2616089), E‐cadherin (BD Biosciences, RRID:AB_397580), αSMA (Dako, Carpinteria, CA, USA, M0851), Mesothelin (Tecan (IBL), Mannedorf, Germany, RRID:AB_2148076), CD169 (Merck Millipore, Burlington, MA, USA, MABT328), p‐PDHE1α (Novus, Centennial, CO, USA, RRID:AB_1291446), GFP (ChromoTek, Planegg‐Martinsried, Germany, RRID:AB_2749857), CD8 (Bioss, Woburn, MA, USA, RRID:AB_10857537)，and F4/80 (Bio‐Rad, Hercules, CA, USA, RRID:AB_2098196).

Techniques: Expressing, Labeling, Membrane, Incubation, Immunofluorescence, Staining, Immunostaining, Fluorescence, Cell Culture, Co-Culture Assay, Western Blot, Injection