Transfection and Nano-CuO Exposure Protocol

Transfection was performed as described in our previous study with modifications [13 (link)]. Briefly, BEAS-2B cells were seeded in the inserts and incubated for 12 h, then U937* cells were seeded at a ratio of 1:1 or 9:1 (BEAS-2B:U937*) onto the BEAS-2B cell layer in antibiotic-free medium supplemented with 10% FBS and allowed to attach. Cells were then transfected with a mixture of 6 µL of TurboFectin 8.0 Transfection Reagent (Origene, Rockville, MD, USA) and 30 nM of MMP-3 siRNA (Ambion, Carlsbad, CA, USA) in a total volume of 1 mL antibiotic-free and FBS-free medium for 6 h. Afterward, 1 mL medium containing 2 times FBS and antibiotics was added, and the cells were incubated for another 12 h. Silencer™ Select Negative Control No. 2 siRNA (Ambion, Carlsbad, CA, USA) was used as a negative control. After exposure to Nano-CuO for 12 h, the conditioned media were collected and the roles of MMP-3 in the cleavage of OPN and activation of fibroblasts were determined.