Fig. 3.
APL cells adhere to ECs, causing impaired barrier function in vitro and in vivo. (A, B) The effect of APL/NB4 cells on ICAM-1 and VCAM-1 expression in HUVECs. Western blots were imaged, and the optical density was calculated using ImageJ. (C) ECs were stained with phalloidin, and RBCs were labeled with live-cell dye. Gap formation in the damaged endothelium permits RBC deposition (arrows). (D) The albumin permeability of the experimental monolayers treated with an adherence receptor antibody. (E) The permeability to RBCs upon treatment with an adherence receptor antibody. (F) The levels of sVCAM-1, sICAM-1, sE-selectin, and sTM in serum samples from the bleeding patient (open circles) and non-bleeding patient (closed circles) groups. (G) Western blotting with an anti-pVE-cadherin antibody, an anti-MLCK, and an anti-ROCK antibody for ECs treated with leukemic cells or supernatant alone. (H) Quantitation of the relative protein expression level in (G) by ImageJ (Gapdh was used as the loading control). (I) Pretreatment with MLCK inhibitor ML-7(10−4 mol/L) or/and with ROCK inhibitor H1152 (2.5 μmol) for 1 h, then incubated with APL cells for 16 h. Permeability of treated endothelium to RBCs. (J) Expression of pVE-cadherin and MLCK in ECs treated with anti-ICAM-1 and anti-VCAM-1 antibodies, respectively, was assayed by western blotting and compared with that in the control and APL samples. (K) Quantitation of the relative protein expression level in (J) by ImageJ. The inset bar represents 20 µm in panel F. AC+APL: Asiatic acid + APL cells. The graph is presented as the mean ± SD of at least five experiments. *P <0.05 and **P <0.01.