Fig. 6.

ANXA2 and hnRNPA2B1 are necessary for the effect of epirubicin on the exosomal export of miR-503 in microvascular endothelial cells. HMVECs were treated with epirubicin for 24 h. Cell lysates were prepared 24 h after treatment and exosomes were collected 72 h after removing the chemotherapeutic drug. Cellular and exosomal levels of miR-503 were evaluated by qPCR. Plot represents mean and SEM (n = 3). b HMVECs were transfected with miR-503-biotin (10 nM). The following day, the cells were crosslinked with DTSSP and UV. HMECs lysates were incubated with streptavidin beads. Both input (IN) (cellular lysate) and pull-down fractions (PD) were separated by SDS-PAGE. Previously identified components of the MEC complex were validated by western blotting against pull-down (PD) and input (IN) (1% of cell lysate) fractions using vinculin as loading control. c HMVECs were transfected with miR-503 (10 nM). 48 h later, immunoprecipitation assays were performed using the indicated antibodies and the levels of miR-503 were evaluated by qPCR. Plot shows fold change of miR-503 in the immunoprecipitated (IP) vs input fractions (IN) in non-treated cells and (d) fold change of immunoprecipitated miR-503 in epirubicin-treated (EPI) vs non-treated cells (NT). Plots represent mean and SEM from three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001. IN = 1% of the cellular lysate before immunoprecipitation