Figure 2. CSE induced CCN1 secretion in lung epithelial cells.

(A) CCN1 secretion induced by CSE (1∼20%). Beas2B cells were treated with CSE in a dose-dependent manner. After 24 h, CCN1 level in the cell supernatant was detected using ELISA. (B) Mouse lung primary type II epithelial cells were exposed to CSE (10 or 20%). After 24 h, CCN1 in cell supernatant was detected as above. (C) Beas2B cells were treated with 10% CSE. After 6 h, ROS was detected using the carboxyl-H2DCFDA methods. (D) CSE induced CCN1 secretion was blocked by anti-oxidant reagents. Beas2B cells were pre-treated with three different anti-oxidant reagents (NAC, ascorbic acid and GSH, 1 mM respectively), followed by 10% CSE. After 24 h, cell supernatant was collected and CCN1 was detected as above. (E) CSE induced CCN1 release was inhibited by ER-Golgi secretion inhibitor, Brefeldin A (BFA), in a dose-dependent manner. Beas2B cells were treated with BFA, followed by CSE. After 24 h, CCN1 secretion was detected using ELISA. (F) Suppression of CSE induced CCN1 secretion by membrane channel protein inhibitors. Beas2B cells were treated with tetraethylammonium chloride (TEA, 100 µM) for aquaporin 1, α18-glycyrrhetinic acid (GA, 100 µM) for connexin, daidzein (50 µM) for caveolin, 2-bromopalmitate (2-BP, 100 µM) for palmitoylation, TGN-020(100 µM) for aquaporin 4. Then these cells were exposed to CSE. After 24 h, CCN1 secretion was determined. For all the above, supernatant from the same amount of cells was collected and subjected to ELSIA. *p<.05 All figures above represented three independent experiments with similar results.