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Blood, Vol. 112, Issue 9, 3624-3637, November 1, 2008
CD40: an upstream master switch for endothelial cell activation uncovered by RNAi-coupled transcriptional profiling
Blood Pluvinet et al.
112: 3624
Supplemental materials for: Pluvinet et al
Files in this Data Supplement:
- Document 1. Supplemental methods and materials (PDF, 108 KB)
- Table S1. Primers used for real-time RT-qPCR (PDF, 59.2 KB)
- Table S2. Gene expression profiling of CD40-regulated genes upon EC stimulation with Jurkat D1.1 (CD154+) T cells (XLS, 2.65 MB)
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List of up-regulated (green) and down-regulated (red) genes fulfilling criteria of B rank ≥ 95 and mean |FC| ≥ 1.2 in at least one of the three stimulation times analyzed: 4, 10 and 16 h.
- Figure S1. Efficient RNAi-mediated CD40 knock-down in ECs (JPG, 39.4 KB)
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HUVECs were electroporated with either siRNA-2 (anti-human CD40) or msiRNA-2 (control), and co-cultured with Jurkat D1.1 (CD154+) cells 4 h prior to analysis, performed at 48 h post-transfection. (A) Human CD40 and ICAM1 mRNA expression levels were assessed relative to the constitutively expressed cyclophilin A gene (ratio of CD40/CYPA or ICAM1/CYPA) by real-time RT-qPCR. Data are presented as means ± SEM from three independent experiments. (B) CD40 receptor levels relative to the ß-actin protein levels were determined by Western immunoblotting. Blot representative of at least three independent experiments. Relative mobility of molecular weight markers is shown in kilodaltons.
- Figure S2. Gene Ontology analysis of the CD40-dependent, Jurkat D1.1 (CD154+)-stimulated EC transcriptome (JPG, 40.2 KB)
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Gene Ontology categories significantly over-represented among the annotated regulated genes (241 genes at the time of analysis) with B rank ≥ 95 and mean |FC| ≥ 1.2 in at least one of the three stimulation times analyzed. (p < 0.05).
- Figure S3. Apelin expression is down-regulated following acute rejection in experimental kidney allotransplantation (JPG, 244 KB)
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Nonrejecting syngeneic transplants were performed between inbred male Wistar-Agouti rats. Rejecting inbred male Wistar-Agouti rats received an allogeneic kidney from Brown-Norway rats. Recipient rats were bilaterally nephrectomized at the moment of transplantation. Animals did not receive any immunosuppressant and were sacrificed at 7 to 10 days post-transplantation. On the day of sacrifice, excised grafts were processed for histological and molecular studies. (A) Total RNA was extracted from both rejecting and non-rejecting kidneys. Rat APLN mRNA expression levels were assessed relative to the constitutively expressed rat 18S ribosomal RNA gene (ratio of rAPLN/18S) by real-time RT-qPCR. Data are presented as means ± SEM from three independent transplantation groups performed in triplicate (*, p < 0.05, versus syngeneic renal transplants). (B) Expression of apelin (arrows) and the endothelial cell marker PECAM-1 (arrowheads) in representative rat kidney graft sections (cortex and medulla) from syngeneic and allogeneic transplants. Control, refers to kidney graft sections from syngeneic transplants in which the primary antibody was substituted by non-immune rabbit serum. Apelin immunoreactivity was evident in the endothelial cells from peritubular and glomerular capillaries and small arterioles from syngeneic kidney grafts, but was almost absent in the allogeneic kidney grafts. In addition, variable apelin immunostaining was present in tubular epithelial cells (with distal tubules appearing more stained than proximal tubules) from non-rejecting kidneys, although a marked decrease in apelin immunoreactivity became also manifest in the rejecting kidneys (×400). Images were taken from a Nikon Eclipse E800 microscope (Nikon Inc., Melville, NY), using a Nikon Plan Fluor 40× / 0.75 objective, a digital SPOT Jr. camera (1.5.0.) (Diagnostic Instruments, Inc., Sterling Heights, MI), and SPOT Advanced imaging software (version 4.0.9).
- Figure S4. Example of enrichment analysis for the up-regulated “NFKB_INDUCED” and “CR_CAM” gene sets at 4 h post-CD40 stimulation, and for the up-regulated “MAP00640_PROPANOATE_METABOLISM” gene set when comparing 4 h versus 16 h post-CD40 stimulation, in HUVECs (JPG, 108 KB)
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The GSEA methodology was used here to analyze molecular profiling data from msiRNA-2-treated versus siRNA-2-treated HUVECs and further stimulated for 4 or 16 h with Jurkat D1.1 (CD154+) cells. (A) Enrichment plots of the up-regulated NFKB_INDUCED, CR_CAM, and MAP00640_PROPANOATE_METABOLISM gene sets. Profile of the running enrichment score (ES), and positions of the gene set members on the rank ordered list for each of the analyzed gene sets. (B) Heat maps of the core-enriched genes in each of the spaces of the NFKB_INDUCED, CR_CAM, and MAP00640_PROPANOATE_METABOLISM gene sets. Blue is up-regulated and pink is down-regulated.
- Figure S5. CD40 engagement through CD154+ T cells induces the anti-viral innate immune surveillance system in ECs (JPG, 63.8 KB)
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Schematic drawing showing key components of the viral immune surveillance system up-regulated upon endothelial CD40 engagement through CD154+ T cells according to the microarray data (Table S2) (a). A further virus challenge facilitating dsRNA recognition by membrane-bound (TLR3) and/or cytosolic (RIG-I, MDA5, OAS3) innate immune response components would induce increased anti-viral (up-regulation of type I interferons, RNA degradation) and pro-inflammatory (up-regulation of CCL2, IL8,…) activities (b).
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