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. 2015 Mar 26;125(22):3377–3387. doi: 10.1182/blood-2015-01-620278

Figure 4.

Figure 4

miR-24 inhibits VWF expression in 293T cells and endothelial cells. (A) Schematic of VWF mRNA and miR-24. miR-24 is partially complementary to a region in the human and mouse VWF 3′UTR. The sequence GAGC in the seed sequences of VWF was mutated to ACTA. (B) miR-24 inhibited luciferase activity of WT VWF 3′UTR but had no effect on luciferase activity of a mutant VWF 3′UTR. The assays were performed in triplicate; ****P < .0001. (C) VWF mRNA levels in cultured HUVEC lysates (harvested 48 hours after transfection with miR-24 mimics, inhibitors, or controls) were analyzed by qPCR (normalized to GAPDH and then normalized to mimic control or inhibitor control). **P < .01. (D) VWF protein levels in cell culture medium (harvested 72 hours after transfection with miR-24 mimics, inhibitors, or controls) were analyzed by ELISA (normalized to the total amount of intracellular protein per well). ***P < .001. (E) VWF mRNA levels in cultured isolated mouse lung endothelial cell lysates (harvested 48 hours after transfection with miR-24 mimics or miR-146a mimics or controls) were analyzed by qPCR (normalized to GAPDH and then normalized to mimic control or inhibitor control). **P < .01; *P < .05. (F) Total VWF protein levels in cell lysates (harvested 72 hours after transfection with miR-24 mimics or inhibitors or controls) were analyzed by ELISA (normalized to mimic control or inhibitor control). ***P < .001; **P < .01. (G) VWF protein level in cell lysates (harvested 72 hours after transfection with miR-24 mimics or controls) were analyzed by western blot; antibodies against CD62P, HSP90, and tubulin were applied as loading controls. (H) VWF protein level in cell lysates (harvested 72 hours after transfection with miR-24 inhibitors or controls) were analyzed by western blot.