Fig. 4

Expression of upstream regulator HDAC5-dependently regulated cellular and exosomal Maspin and anti-angiogenic function caused by human recombinant Maspin. HDAC5 siRNA was transfected into HepG2 in a dose-dependent manner, and after 24 h, HDAC5, p53, Maspin, and TSAP6 expressions were analyzed by western blot. The expression of each protein was quantified using b-actin as an internal control (A). After HDAC5 siRNA was transfected into HUVECs, Maspin mRNA expression was investigated. It was confirmed using the GSE15499 GEO data set (B). Data are shown as mean ± SD of three independent values. *P < 0.05 vs. con siRNA by Student’s t-test. HepG2 was exposed to 4 Gy RT 24 h after HDAC5 siRNA was transfected. After overnight, the expression of HDAC5, Maspin, HSP90, and Calnexin in cell lysates and exosomes of each group was examined by western blot (C, D). HUVECs were seeded on growth factor-reduced matrigel-coated plates and treated with VEGF (50 ng/ml), human recombinant Maspin (1-10ug/ml). After 16 h, calcein am dye (0.5ug/ml) was treated and fluorescence images were photographed using EVO M5000 Imaging System (Invitrogen) (E). Five locations of each well were randomly photographed, and the branching and tube organization were measured and compared with that of the control condition (F, G). Cell culture dishes were performed in at least three separate experiments. Scale bar = 200 μm. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control by Student’s t-test