NS1 induces MMP-9 secretion from human leukocytes but not HUVECs.

<p><b>(A)</b> HUVECs were treated with PBS or NS1 for the indicated times; then, the supernatants were collected for MMP-9 detection by ELISA. (n = 3) <b>(B)</b> Isolated human leukocytes (WBCs) and <b>(D)</b> PMA-activated THP-1 cells were treated with PBS or NS1, and the culture medium was collected at the indicated times. The concentration of MMP-9 in the culture medium was determined by ELISA. (n = 4) <b>(C)</b> Isolated WBCs were treated with PBS, NS1, or NS1 mixed with anti-NS1 antibodies for 24 h, and the concentration of MMP-9 in the culture medium was determined by ELISA. (n = 4) <b>(E)</b> After PMA activation, THP-1 and primary isolated WBCs were subjected to the desired treatment. After 24 h, the cell culture supernatants were collected. An MMP antibody array that detects various MMPs and TIMPs was used to assess the major subclass of MMPs induced by NS1. THP-1 cells or WBCs were stimulated with PBS or NS1; then, the culture supernatants were collected and analyzed for extracellular matrix proteins. Membranes of the human MMP antibody array were probed with the supernatant collected from bovine serum albumin (BSA)-treated THP-1 cells, BSA-treated WBCs, 20 μg/ml NS1-treated THP-1 cells or NS1-treated WBCs. The quantification of MMPs array membranes was analyzed by ImageJ. PC, positive control; NC, negative control. <b>(F)</b> After treatment with 20 μg/ml NS1 for 24 h, the 5X-concentrated WBCs and 10X-concentrated THP-1 supernatants were analyzed by electrophoresis with a 7.5% acrylamide gel containing gelatin. The gel was stained with Coomassie blue to reveal the white bands corresponding to the proteolysis of gelatin by MMPs. S/N, supernatant; *P<0.05, **P<0.01, ***P<0.001; unpaired t-test (panel B and D), Kruskal-Wallis ANOVA (panel C).