Figure 5
Figure 5. Platelet MP-derived miR-223 can regulate HUVEC gene expression at the mRNA level. (A) HUVEC transiently expressing a Rluc reporter gene, harboring the 3′UTR of FBXW7 (pRL-CMV-3′UTR FBXW7) (left panel) or EFNA1 mRNA (pRL-CMV-3′UTR EFNA1) (right panel), were incubated (or not; control) with MPs derived from thrombin-activated platelets for 48 hours prior to luciferase activity measurements. Results were normalized on Fluc activity, and expressed as mean (±SEM) percentage of control (n = 3 experiments). (B-C) HUVEC transiently expressing a miR-223 sponge (pRL-CMV-BS miR-223 vector; n = 3 experiments) (C), or not (n = 5 experiments) (B), were incubated with platelet-derived MPs for up to 48 hours, and FBXW7 (left panels) and EFNA1 (right panels) mRNA levels were quantitated by qPCR. Results were normalized by the 2^-ΔΔCt method, using glyceraldehyde-3-phosphate dehydrogenase mRNA as a reference, and expressed as mean (±SEM) fold changes vs baseline. *P < .05; **P < .01; ***P < .0001 vs control or baseline (Student t test).

Platelet MP-derived miR-223 can regulate HUVEC gene expression at the mRNA level. (A) HUVEC transiently expressing a Rluc reporter gene, harboring the 3′UTR of FBXW7 (pRL-CMV-3′UTR FBXW7) (left panel) or EFNA1 mRNA (pRL-CMV-3′UTR EFNA1) (right panel), were incubated (or not; control) with MPs derived from thrombin-activated platelets for 48 hours prior to luciferase activity measurements. Results were normalized on Fluc activity, and expressed as mean (±SEM) percentage of control (n = 3 experiments). (B-C) HUVEC transiently expressing a miR-223 sponge (pRL-CMV-BS miR-223 vector; n = 3 experiments) (C), or not (n = 5 experiments) (B), were incubated with platelet-derived MPs for up to 48 hours, and FBXW7 (left panels) and EFNA1 (right panels) mRNA levels were quantitated by qPCR. Results were normalized by the 2^-ΔΔCt method, using glyceraldehyde-3-phosphate dehydrogenase mRNA as a reference, and expressed as mean (±SEM) fold changes vs baseline. *P < .05; **P < .01; ***P < .0001 vs control or baseline (Student t test).

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