cd36 blocking antibody sr bi  (Novus Biologicals)

Journal: PLOS Pathogens

Article Title: Macrophage MST1 protects against schistosomiasis-induced liver fibrosis by promoting the PPARγ-CD36 pathway and suppressing NF-κB signaling

doi: 10.1371/journal.ppat.1012790

Figure Lengend Snippet: PEMs and liver tissues were collected from Mst1 +/+ and Mst1 ΔM/ΔM mice infected with 20 ± 5 cercariae and sacrificed at week 12. (A) RNA-seq analysis was performed on differentially expressed genes (DEG) in PEMs from infected Mst1 +/+ and Mst1 ΔM/ΔM mice control Mst1 +/+ and Mst1 ΔM/ΔM mice (n = 3). DEGs were identified in all pairwise comparisons and showed a 2-fold change in expression with an adjusted p- value of 0.05. (B) Some DEGs were verified by qPCR in PEMs from infected mice. (C) Liver sections from infected mice were subjected to HE staining (leftmost panel) and dual immunofluorescence for CD36 and F4/80 (2–5 panels on the right side) in. High-magnification images of co-staining for F4/80 and CD36 cells were shown in the right most panel. The fluorescence intensity of CD36 overlaid with F4/80 + cells was analyzed using ImageJ. Data are shown as mean ± SEM of three different liver sections in each mouse (n = 9, data pooled from 3 independent experiments, 3 mice per group for each experiment. Bar of the left five panels = 100 μm, bar of the far right panel = 50 μm).All p values were determined by Student’s t -test. * P < 0.05, ** P < 0.01.

Article Snippet: The CD36 blocking antibody SR-BI (NB400-104) was purchased from Novus Biologicals (Littleton, Colorado, USA).

Techniques: Infection, RNA Sequencing, Control, Expressing, Staining, Immunofluorescence, Fluorescence

Journal: PLOS Pathogens

Article Title: Macrophage MST1 protects against schistosomiasis-induced liver fibrosis by promoting the PPARγ-CD36 pathway and suppressing NF-κB signaling

doi: 10.1371/journal.ppat.1012790

Figure Lengend Snippet: WT or Mst1 deficiency peritoneal exudate macrophages (PEMs) were stimulated with or without SEA (50μg/ml) for 24h, followed by incubation with FITC-Dextran for 2 h. (A) Flow cytometry analysis of the proportion of CD36 + cells. Isotype-matched control IgG staining (----); anti-mouse CD36 staining (——, no SEA;——, with SEA) The representative percentage of CD36 positive cells are shown in the left panels. Gating strategies are shown in the left panels. (B) Flow cytometry analysis of the proportion of PEMs engulfing FITC-dextran, with analysis of mean fluorescence intensity (MFI) in engulfed PEMs. (C) Immunofluorescence assay of PEMs engulfing FITC-dextran, with representative images from three separate cell isolates (magnification×200) and percentages of FITC + in F4/80 + PEMs shown. Ten viewing fields were analyzed in each experiment(Bar of the left three panels = 40 μm, bar of the far right panel = 20 μm). (D, E) Changes in gene expression in PEMs after the uptake of FITC-dextran (37°C, 2h). (F, G) SEA cultured WT PEMs were pretreated with isotype control (clone EPR25A, ab172730) and or CD36 function-blocking antibody (clone EPR22509-40, ab252922), followed by incubation with FITC-Dextran (2 h, 37°C), and subjected to immunofluorescent staining analysis. Representative images were shown (magnification×200) on left panels. Percentages of coimmunostaining of CD36 + and FITC + PEMs were shown on the right side (Bar of the left three panels = 40 μm, bar of the far right panel = 20 μm) (F). mRNA expressions of indicated genes were detected by qPCR (G). Data are shown as mean ± SEM (n = 3, three samples consisting of pooled PEMs of two or three mice from representative of three independent experiments). Statistical significance was determined by student t test (A-F) and one-way ANOVA analysis (G). * P < 0.05, ** P < 0.005, *** P < 0.001.

Article Snippet: The CD36 blocking antibody SR-BI (NB400-104) was purchased from Novus Biologicals (Littleton, Colorado, USA).

Techniques: Incubation, Flow Cytometry, Control, Staining, Fluorescence, Immunofluorescence, Gene Expression, Cell Culture, Blocking Assay

Journal: PLOS Pathogens

Article Title: Macrophage MST1 protects against schistosomiasis-induced liver fibrosis by promoting the PPARγ-CD36 pathway and suppressing NF-κB signaling

doi: 10.1371/journal.ppat.1012790

Figure Lengend Snippet: (A) Pathway analysis of the RNA-Seq data comparing PEMs from Mst1 +/+ and Mst1 ΔM/ΔM mice infected by S . japonicum for 12 weeks. (B) Immunofluorescence assay of SEA (24 h, 50μg/ml) or control cultured WT and MST1- deficiency PEMs for CD36 and PPARγ. Images were collected with a confocal microscope and CD36 intensity was quantified using ImageJ. Data are shown as mean ± SEM (n = 3, three samples consisting of pooled PEMs of two or three mice from representative of three independent experiments. Bar = 10 μm). (C) Immunofluorescence assay for CD36 (green), PPARγ (red) in RAW264.7 cells stimulated with or without SEA (24 h, 50μg/ml), followed by XMU-MP-1 incubation (3μM, 6h). Images were taken with a confocal microscope. Percent nuclear PPARγ fluorescence and CD36 intensity in macrophages with PPARγ localization were analyzed using ImageJ. Data from at least three independent experiments, with 10 viewing fields were analyzed in each (Bar of the left four panels = 10 μm, bar of the far right panel = 5 μm). (D, E) Immunoblot analysis of phosphorylated (p-)PPARγ and total PPARγ (left and middle) and relative Cd36 mRNA (right) in RAW264.7 cells treated with SEA ± XMU-MP-1(D) and in liver tissues from Mst1 +/+ and Mst1 ΔM/ΔM mice infected and control mice (E). Values are means ± SEM (n = 3, from representative of three independent experiments). Statistical significance was determined by Mann-Whitney U-test (B), one way ANOVA analysis (C) and Student’s t test (D-E). * P < 0. 05, ** P < 0. 01.

Article Snippet: The CD36 blocking antibody SR-BI (NB400-104) was purchased from Novus Biologicals (Littleton, Colorado, USA).

Techniques: RNA Sequencing, Infection, Immunofluorescence, Control, Cell Culture, Microscopy, Incubation, Fluorescence, Western Blot, MANN-WHITNEY