Fig 2: The role of MMP-3-cleaved OPN in Nano-CuO-induced activation of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at the ratio of 1:1 (a and b) or 9:1 (c and d) (BEAS-2B:U937*) in the inserts, and MRC-5 fibroblasts were seeded in the lower chambers of Transwell® units. MRC-5 fibroblasts were pretreated with 200 µg/mL of GRGDSP peptides, then BEAS-2B and U937* cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts were incubated for another 36 h. The proteins from MRC-5 fibroblasts were isolated to determine the activation of MRC-5 fibroblasts by Western blot. a and c were the results of a single Western blot experiment. b and d were the average expression levels of a-SMA, Col1A1, and fibronectin normalized to ß-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated alone group, p < 0.05

Fig 3: Schematic diagram of potential mechanisms of Nano-CuO-induced activation of fibroblasts. Exposure of lung epithelial BEAS-2B cells and U937* macrophages to Nano-CuO caused increased expression and activity of MMP-3 and increased production of MMP-3-cleaved OPN, leading to the activation of fibroblasts. Graphics were created with BioRender (https://BioRender.com)

Fig 4: The role of MMP-3 on Nano-CuO-induced production of cleaved OPN in BEAS-2B cells and U937* macrophages. BEAS-2B cells (a and b) and U937* macrophages (c and d) were transfected with 30 nM of MMP-3 siRNA or Negative Control No. 2 siRNA. After transfection, the cells were exposed to 1 µg/mL of Nano-CuO for 12 h. The conditioned media were collected to detect the level of cleaved OPN (40 kDa N-terminal fragment) by Western blot. Equal protein loading was verified by Coomassie Brilliant Blue staining. a and c were the results of a single experiment. b and d were normalized band densitometry readings averaged from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated group transfected with Negative Control No. 2 siRNA, p < 0.05

Fig 5: Increased expression of OPN in BEAS-2B cells and U937* macrophages exposed to Nano-CuO. BEAS-2B cells, U937* macrophages, or co-culture of BEAS-2B and U937* macrophages at a ratio of 1:1 or 9:1 (BEAS-2B:U937*) were exposed to 0.5 and 1 µg/mL of Nano-CuO for 12 h. After treatment, the cells and conditioned media were collected. Cells without treatment were used as control. Full-length OPN (66 kDa) in the cells and cleaved OPN (40 kDa N-terminal fragment) in the conditioned media were detected by Western blot. Equal protein loading was verified by ß-actin expression (a and b) or Coomassie Brilliant Blue staining (c and d). a and c were the results of a single experiment. b and d were results of three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05