Fig. 9

Indirect exposure to Nano-CuO caused activation of MRC-5 fibroblasts in a triple co-culture system. a is the experimental protocol for the triple co-culture experiments. BEAS-2B cells and U937* cells were seeded at the ratio of 1:1 (b and c) or 9:1 (d and e) (BEAS-2B:U937*) in the inserts of Transwell^® units and MRC-5 cells were seeded in the lower chambers of the units. After establishment of the triple co-culture system, BEAS-2B and U937* cells were exposed to 0, 0.5 and 1 µg/mL of Nano-CuO for 12 h, then the inserts were taken out. MRC-5 cells in the lower chamber were incubated for another 36 h and proteins were isolated to detect the expression of α-SMA, Col1A1, and fibronectin. b and d were the results of a single Western blot experiment. c and e were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05. Graphics in a were created with BioRender (https://BioRender.com)