Journal: bioRxiv

Article Title: An evolutionary transcriptomics approach links CD36 to membrane remodeling in replicative senescence

doi: 10.1101/294512

Figure Lengend Snippet: Characterization of genes associated with replicative senescence. (A) The cumulative fraction plot of pLI (the probability of being loss-of-function intolerant) value of genes upregulated in old cells (YO, blue) and other protein-coding genes (gray). This measurement is obtained from ExAC database . (B) The cumulative fraction plot of b-value (the index of background selection between primate species) of genes in each category (Mc ). The color scheme is same as the previous panel. (C) X-axis shows the tolerance of genes in healthy human genomes (-log p- value of pLI), y-axis shows the log 2 fold change between young and old in BJ cells and the size of the bubble indicates the log 2 fold change between young and old in MRC-5 cells. The strength of the color shows the -log p-value of pLI. CD36 showed upregulation in both old BJ and MRC-5 cells and tolerance to loss-of-function mutations in healthy human genomes.

Article Snippet: FusionRed-CD36-C10 plasmid (#56104) was obtained from Addgene (deposited by the Davidson lab).

Techniques: Selection

Journal: bioRxiv

Article Title: An evolutionary transcriptomics approach links CD36 to membrane remodeling in replicative senescence

doi: 10.1101/294512

Figure Lengend Snippet: CD36 overexpression results in senescence-like phenotype. (A) Representative images of control and CD36 -overexpressing young MRC-5 cells. CD36 o verexpression was visualized via the FusionRed tag (scale bar = 10µm). (B-C) Measurement of β-galactosidase activity in control and CD36 overexpressing MRC-5 cells. A significant increase of SA-β-galactosidase positive cells was observed during CD36 overexpression for which representative images are shown (scale bar =100µm for control and center CD36 images, and 20µm for furthest to the right CD36 image). Data shown as means ± standard deviation, *** p < 0.001. (D) Representative western blot showing p16 levels in control and CD36 overexpressing young MRC-5 cells.α-tubulin was used as a loading control. We observe a modest increase in p16 levels. (E) Measurement of β-galactosidase activity in young MRC-5s that were treated with conditioned media. “Control” represents cells in standard growth medium, “Control_M” represents cells in medium from control wells during transfection, “+CD36_M1” represents cells in medium from CD36 overexpressing cells 48h after transfection, and “+CD36_M2” represents cells in medium from CD36 overexpressing cells 96h after transfection. An increase in SA-β-galactosidase positive cells was observed.

Article Snippet: FusionRed-CD36-C10 plasmid (#56104) was obtained from Addgene (deposited by the Davidson lab).

Techniques: Over Expression, Activity Assay, Standard Deviation, Western Blot, Transfection

Journal: PLoS ONE

Article Title: Plasmodium falciparum FIKK Kinase Members Target Distinct Components of the Erythrocyte Membrane

doi: 10.1371/journal.pone.0011747

Figure Lengend Snippet: A. The adhesion phenotypes of the two KO parasite lines were investigated. The FIKK7.1- and FIKK12-KO parasites and wild type FCR3 were selected on cells expressing either CSA (white bar) or CD36 (black bar), after 3 rounds of selection all the three parasite lines showed similar binding densities on BeWo (CSA) or CHO745-CD36 cells. B. Cellular localization of A-type Rifin, Stevor and Surfin 4.2 in FIKK-KO and wild type parasites. Antigen was detected using specific anti-Rifin, Surfin or Stevor antibodies by immunofluorescence assay.

Article Snippet: Briefly, plastic Petri dishes were coated overnight at 4°C with phosphate-buffered saline (PBS) containing 1 mg/ml CSA sodium salt from bovine trachea (Sigma), 1 mg/ml chondroitin sulfate C sodium salt from shark cartilage (Sigma), 10 μg/ml recombinant human CD36 (R&D Systems).

Techniques: Expressing, Selection, Binding Assay, Immunofluorescence

Journal: Clinical and Experimental Immunology

Article Title: Investigating the role of proinflammatory CD16 + monocytes in the pathogenesis of inflammatory bowel disease

doi: 10.1111/j.1365-2249.2010.04177.x

Figure Lengend Snippet: The peripheral blood CD14+ CD16+ monocyte population is increased in active Crohn's disease. (a) Peripheral blood mononuclear cells (PBMC) were stained for CD14, CD16 and CD36 and analysed by flow cytometry. Cells were pre-gated for high forward-scatter (FSC), side-scatter (SSC) and CD36 expression, and monocytes separated by CD14/CD16 expression. The lower panels show representative dot plots from a healthy volunteer, and Crohn's disease patients with quiescent [Crohn's disease activity index (CDAI)<150] and active (CDAI>150) inflammation. (b) The ratio of CD14+ CD16+ monocytes was increased significantly in Crohn's disease patients compared to healthy controls. *P = 0·01 (two-tailed Student's t-test with Welch's correction). (c) Further categorization of patients revealed that the CD14+ CD16+ population was increased in active Crohn's disease compared to healthy controls, but not quiescent disease or patients with ulcerative colitis. *P < 0·05 (Bonferroni's post-test following one-way analysis of variance).

Article Snippet: Slides were then incubated at 4°C overnight with the following primary antibodies: mouse anti-CD14 (BioLegend, San Diego, CA, USA), rat anti-CD16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-CD36 (Novus Biologicals, Littleton, CO, USA).

Techniques: Staining, Flow Cytometry, Expressing, Activity Assay, Two Tailed Test

Journal: Clinical and Experimental Immunology

Article Title: Investigating the role of proinflammatory CD16 + monocytes in the pathogenesis of inflammatory bowel disease

doi: 10.1111/j.1365-2249.2010.04177.x

Figure Lengend Snippet: The number of CD14+ CD16+ monocytes is enhanced significantly in the lamina propria of Crohn's disease patients. (a) Monocytic lineage of CD14+ CD16+ cells in the colonic mucosa was confirmed by three-channel immunofluorescence microscopy for CD14 (green), CD16 (red) and CD36 (blue). No or limited CD36 staining was observed in CD14+ CD16– cells (i) and CD14– CD16+ cells (ii). In contrast, CD14+ CD16+ cells stained strongly for CD36 (iii, white pseudocolour). Colonic crypts are indicated with dashed lines. Lower panels, magnification of the boxed cells shown above. (b) Representative images of colonic mucosal sections from non-inflammatory bowel disease (IBD) control patients, non-inflamed and inflamed tissue from Crohn's disease patients, and non-inflamed ulcerative colitis samples. CD14+ CD16+ monocytes are shown in yellow pseudocolour, with nuclei in blue. (c) The number of CD14+ CD16+ cells per high-power field (HPF) was significantly increased in Crohn's disease mucosa, and enhanced further in actively inflamed tissue. Each data point represents the average of five or more HPF per patient sample. *P < 0·001 versus control, †P < 0·001 versus IBD – not inflamed [Bonferroni's post-test following one-way analysis of variance (anova)]. (d) The ratio of CD14+ CD16+ cells was increased significantly in Crohn's disease patients compared to controls. *P < 0·001 (Bonferroni's post-test following one-way anova). (e) The increase in total CD14+ CD16+ monocytes was confirmed by flow cytometry. Mononuclear cells were isolated from control and Crohn's disease colonic mucosa as described in the Materials and methods, pre-gated for forward-scatter (high), side-scatter (high) CD36+, and separated by CD14/CD16 expression. A dramatic increase of CD14+ CD16+ monocytes was seen in IBD tissue (red box). Plots are representative of three patient samples per group. (f) The tumour necrosis factor (TNF)-α mean fluorescence intensity (MFI, arbitrary units) of CD14+ CD16+ monocytes was significantly higher compared to CD14+ CD16– cells in the same field of vision. The graph shows the result from 14 HPF taken from three Crohn's disease patient samples. *P < 0·001 (two-tailed Student's t-test).

Article Snippet: Slides were then incubated at 4°C overnight with the following primary antibodies: mouse anti-CD14 (BioLegend, San Diego, CA, USA), rat anti-CD16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-CD36 (Novus Biologicals, Littleton, CO, USA).

Techniques: Immunofluorescence, Microscopy, Staining, Flow Cytometry, Isolation, Expressing, Fluorescence, Two Tailed Test