Transwell membrane

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 used a Transwell membrane (8 µm pore size; BD Biosciences, Bedford, MA, USA) coated with Matrigel (BD Biosciences) for invasion assay. Cells were grown to 60–80% confluence after trypsinization. Cells (2 × 10⁵, 100 μL) in the serum-free RPMI-1640 were put into the upper chamber of each well of a 24-well transwell polycarbonate membrane (8 µm pore size) coated with 50 µL Matrigel. The RPMI-1640 medium (600 μL) containing 10% FBS served as a chemoattractant, was put into the lower chambers. After wells were incubated for 24 h at 37°C, the non-invaded cells in the upper compartment were removed and the chamber was washed twice with PBS. The cells that had invaded through the membrane were stained with methanol and 0.1% crystal violet, imaged, and counted using an inverted microscope (Olympus, Tokyo, Japan) and quantified from visualizing five random fields at a magnification of ×400

In vitro injury was given for 6 h by treating cultured hepatocytes with a 5 mM concentration of CCl₄ in DMSO, as described previously [27 (link)]. The co-cultured model was established by culturing injured hepatocytes treated with MSCs and compounds (18), (14) and (10) for 24 h in a Transwell culture system with DMEM (sigma) medium containing 10% FBS, 100 μg/mL of streptomycin and 100 U/mL of penicillin. The co-culture was undertaken using a porous Transwell membrane, with a pore size of 0.4 mm (BD Biosciences). Hepatocytes were first seeded at a density of 1.5 × 10⁵/cm² on the collagen-coated 6-well plate. Once attached, the MSCs were seeded onto the Transwell membrane inserts at a density of 1.5 × 10⁴. The hepatocytes were divided into six groups: normal, CCl₄-treated injured, CCl₄-treated injured co-cultured with MSCs alone, CCl₄-treated injured co-cultured combined with MSCs + compound (18), CCl₄-treated injured co-cultured combined with MSCs + compound (Luciferin Generation Reagent) and CCl₄-treated injured co-cultured combined with MSCs + compound (10). After 24 h of co-culturing, the treated hepatocytes cells were then collected for the extraction of their RNA, LDH cytotoxicity assay, trypan blue assay, glutathione assay and immunostaining.

Migration assays were performed using Transwell membranes (Corning, New York, USA). After 48 h of infection, the cells (1×10⁵) were plated in the upper chamber with serum-free medium, while the lower compartment was filled with 20% FBS as a chemo attractant. After incubation for 24h, the cells that remained in the upper chamber were carefully removed with cotton swabs, while the cells that had migrated were fixed in 3% paraformaldehyde and stained with crystal violet. Cells in 5 independent 20x fields per well were then counted and imaged under a microscope.
For the invasion assay, prior to the inoculation of cells in the Transwell upper chamber, Transwell membranes were coated with Matrigel (BD-Bio, USA). Otherwise, the experimental process was performed as described above.

For cell invasion assay, transwell membranes (BD, Franklin Lakes, NJ, USA) were precoated with Matrigel. After transfection for 24 h, SGC7901 cells were resuspended in serum-free RPMI 1640 medium with a density of 1 × 10⁶/ml. The cell solution of 100 μl was added into the upper transwell chamber, and the lower chamber was added with 500 μl of 20% FBS-containing RPMI 1640 medium. After 48 h of incubation, the cells on the upper surface of transwell membranes were removed, and invasive cells on the lower surface were stained with crystal violet for 10 min. Positive stained cells were counted under a microscope (Olympus) at five random fields.
For wound healing assay, transfected cells were cultured in complete RPMI 1640 medium to 90% confluence. After that, the cell monolayers were scratched with a 10 μl pipette tip to create a single-line wound. The cells were washed by PBS to remove suspended cells. Next, the cells were cultured in the serum-free medium at 37°C with 5% CO₂ for 48 h. The wound area at 0 h and 48 h was observed using a microscopy and quantified using ImageJ software.