Ckx53

Parts of our experiments were performed on the human bronchial epithelial cell line (16HBE14σ, MERCK, Darmstadt, Germany). Cells were cultured on T75 flasks (Nunc, Thermo Fisher) in Minimal Essential Medium (MEM; MERCK, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and antibiotics—100 U/mL penicillin and 100 mg/mL streptomycin (MERCK, Darmstadt, Germany). Cells were maintained within a humidified 5% CO₂ atmosphere at 37 °C and reseeded when they reached 90% confluence.
To illustrate the effect of particulate matter (PM) on the human bronchial epithelium (16HBE14σ), the cells were grown in cell culture medium alone (control) or supplemented with PM. Cell morphology was viewed after 24, 48 and 72 h of incubation using an inverted optical microscope (Olympus CKX53, Olympus, Tokyo, Japan).

The HGFs were seeded at a concentration of 1 × 10
⁵cells/well in 500 µL of DMEM containing 10% FBS in a 24-well plate and incubated at 5% CO
₂and 37°C for 24 hours. After that, a single layer of HGFs was scraped with a P1000 pipette tip at the center of the well plate. The medium was removed and washed twice with PBS. HGFs were treated with 500 µL of PDLSC-CM at concentrations of 25 and 50 µg/mL. HGFs treated with DMEM with 10% FBS were used as controls. The plate was placed under an inverted microscope (Olympus CKX53, Shinjuku-ku, TYO Japan) using 4X magnification to take photos of the gap before the experiment. Cell migration was observed within 24 to 48 hours. Then, the medium was removed and washed with PBS twice per well. The HGFs were fixed in 100% methanol (Qrec, New Zealand) for 2 minutes before being rinsed twice with PBS. Then, 10% Giemsa stain (Merck) was added to stain HGFs for 10 minutes and rinsed with PBS twice. Photos were taken of the gap after the experiment. For each image, the HGFs that migrated to the created gap were counted by ImageJ software. The percentage of cell migration was calculated as follows:

For wound healing migration assay, A549 and H460 cells (1 × 10⁵) were seeded into each compartment of the culture insert (Ibidi, Martinsried, Germany). A cell-free gap of 500 μm was created after removing the insert, and the cells were treated with ZVI-NPs during the migration process. For transwell invasion assay, 5 × 10⁵ cells were seeded onto the upper side of transwell membrane (Falcon) which was precoated with Matrigel (Corning, New York, NY, USA) one day before seeding the cells. After ZVI-NP treatment for 16 h, the cells attached on the reverse side of the membrane were fixed and stained. Random views of both migration and invasion assay were photographed by Olympus CKX53 (Olympus, Tokyo, Japan) and analyzed by ImageJ software.

We cultured 1 × 10⁵ BMMs per well in 24-well hydroxyapatite-coated plates (Corning Life Sciences, St. Lowell, MA, USA) and treated with 0, 10, or 100 μM puerarin in α-MEM medium containing with 50 ng/mL RANKL and 50 ng/mL M-CSF for 5 days. Then, the wells were washed with PBS to remove cells, and the areas of hydroxyapatite resorption was observed by a Optical Microscope Olympus CKX53 (Tokyo, Japan) and quantified with the Image J software (NIH, Bethesda, Maryland, USA).

Cells (2 × 10⁴ cells/mL) were seeded into 96-well plates and were incubated at 37 °C and 5% CO₂ for 24 h. Viruses were incubated with Cu MPs or Cu NPs suspensions as described in Section 2.7 for indicated times and then added to cells for infection. Twenty-four hours after infection, 10 µL Cell Counting Kit-8 (Dojindo, Rockville, MD, USA) reagent was added to each well and the cells were incubated for 1 h at 37 °C. The optical density was measured at 450 nm by a microplate spectrophotometer. Morphological characterization was analyzed using inverted microscope Olympus CKX53 (Olympus, Tokyo, Japan) with different magnifications and digitally visualized using software iSolution Auto plus (IMT Inc., Daejeon, Korea). For the evaluation of Cu MPs and Cu NPs cytotoxicity, Cu MPs and Cu NPs were first dispersed in PBS (5% w/v), added to cells to the final concentration of 0.5% w/v, and incubated for the indicated time.