Fig. 5

Anti-inflammatory effects of ENO1 Ab HL217 in bleomycin-stimulated primary human endothelial cells (HUVEC). Primary human umbilical vascular endothelial cells (HUVEC) were treated with 50 ng/ml bleomycin (BLM) for 24 h and subjected to the measurement of surface ENO1 expression (A, B) by using immunofluorescence staining. (A) Representative pictures of 4 independent experiments were shown. Cells expressing surface ENO1 were indicated with white arrowhead in the upper panel (ENO1 expression shown in green). The lower panel (merged with nuclear staining DAPI to indicate location of all cells) was kept without arrowhead for clarity. Cells expressing surface ENO1 were manually counted in 5 area of one high power field picture. Four pictures of each group were taken and counted. (B) Percentages (%) of surface ENO1 expressing cells was determined over the number of all nucleated cells. (C-E) HUVEC were treated with indicated concentrations of BLM, HL217, hIgG1, and plasmin inhibitor tranexamic acid (TXA) for 24 h. Cell-associated plasmin activity was measured (C) and cell culture supernatants were collected for measurement of chemokines, including CCL2 (D) and IL-8 (E), by ELISA. Scale bar, 100 µM. ^#P < 0.05, ^###P < 0.001 vs. untreated cells; *P < 0.05, **P < 0.01, ***P < 0.001 vs. the BLM-treated group. Pooled data of three independent experiments were shown as mean ± SD (one technical replicate and three biological repeats per group)