Figure 4

miR-126 shuttled by exosomes modulate CXCL12 expression in HUVECs. a: Real time PCR analysis showed that CXCL12 mRNA expression decreased in dose-dependent (20 and 50 μg/ml) manner after addition of exosomes to endothelial cells. Values are the mean ± SD of 3 independent experiments **p ≤ 0.01. b: CXCL12 protein levels, assessed by ELISA, in HUVEC-conditioned medium obtained after 24 hours of stimulation with: low serum medium (Control), 20 μg/ml of exosomes (Exo 20 μg/ml) and 50 μg/ml of exosomes (Exo 50 μg/ml). Values are the mean ± SD of 3 independent experiments **p ≤ 0.01. c: Confocal microscopy analyses of HUVECs treated with 50 μg/ml of exosomes for 24 h. HUVECs were stained with Texas Red-conjugated anti-CXCL12 antibodies, nuclear counterstaining was performed using Hoescht (blue). Scale bar = 10 μm. d: CXCL12 protein levels, assessed by ELISA, in HUVEC-conditioned medium obtained after 24 hours of stimulation with: low serum medium (Control), 20 μg/ml of exosomes (Exo 20 μg/ml) and 50 μg/ml of exosomes (Exo 50 μg/ml). CXCL12 protein levels were also evaluated in HUVECs transfected with miR-126 inhibitor (2-O-Me-miR-126) and treated with: 20 μg/ml of exosomes (Exo 20 μg/ml + 2-Me-miR-126) and 50 μg/ml of exosomes (Exo 50 μg/ml + 2-Me-miR-126). As a negative control, miScript Inhibitor Negative Control (Scramble) was used. e: CXCL12 protein levels were also evaluated in HUVECs transfected with miR-126 mimic and treated with: 20 μg/ml of exosomes (Exo 20 μg/ml + miR-126 mimic) and 50 μg/ml of exosomes (Exo 50 μg/ml + miR-126 mimic). As a negative control of transfection, the AllStars Negative Control (Scramble) was used.