Silicone insert

The in vitro wound healing migration assay was optimized according to Ho et al. [28 ]. Wound healing and migration assays were done by seeding the 786-O cells (3.5 × 10⁴/well) into the Ibidi-silicone insert (Ibidi; Cat. #81176, Germany) and incubated for attachment. This insert allows the formation of a well‐defined edge without physically scratching or wounding the cell monolayer after removal of the inserts. In order to monitor cell movement within the wounded area following their incubation with diverse concentrations of cisplatin combined with HWG or RA, three wounds were photographed immediately after picking up the insert (T₀) and after 6 h. The endpoint of the assay was measured by calculating the reduction in the width of the wound after 6 h and comparing it to T₀ with the non-treatment control group set at 100%.

RT4 cells were first seeded at a concentration of 4 × 10⁴ cells/100 μl in both chambers of an Ibidi-silicone insert (Ibidi, Martinsried, Germany). This insert allows for the formation of a well-defined edge without physically scratching or wounding the cell monolayer. Cells were cultured for 24 h in DMEM containing 10% FBS to form a confluent monolayer. Then, cells were grown in serum-free DMEM and Mitomycin C (5 μg/ml, Sigma #M4287) to inhibit cell proliferation for 12 h prior to careful removal of the insert. Cells were incubated in the CM from HFs, HFs + TGFß or iCAFs for 24 h in presence or absence of an anti-IL-6 antibody (1 μg/mL). The migration was visualized at the indicated times (0, 6, and 12 h) under an inverted microscope (TE2000, Nikon). Migration distances were measured using the ImageJ analysis software (National Institutes of Health, Bethesda, MD).

Differentiated iBMECs were purified on CN IV-FN and subsequently subcultured onto 2-well plates with silicone inserts (ibidi) designed for the wound healing assay. The wells were pre-coated with LN 511-E8 or CN IV-FN. The next day, the inserts were removed and the medium was changed to endothelial cell medium without bFGF and retinoic acid. Using an EVOS FL Auto microscope, phase contrast images were taken every 3 h from the same spot, and the area void of cells in each image was calculated using ImageJ. Three independent replicates per condition were evaluated for this experiment.

P0 rat cardiac fibroblasts were used for cell scratch/wound healing assays. Silicone inserts (Ibidi, Martinsried, Germany) were placed in a 6-well culture dish and a cell suspension of 2.85 × 10⁹ cells/mL was prepared. 70 μL of this cell suspension (approximately 2.5 × 10⁵ cells) was added to each chamber of the silicone insert separated by the silicone divider of the insert. The area surrounding the insert was covered with 1.5 mL DMEM-F12 media to prevent drying out of cells. Cells were allowed to adhere and grow overnight in complete growth media (10% FBS DMEM-F12). The next day, the media was changed to a 2% FBS DMEM-F12 solution and cells were incubated in low serum for an additional 24 hours. Cells were then treated with Baf-A1 (7.5 nM) or CQ (35 μM) for 48 hours. After 48 hours, inserts were removed and light microscopy images acquired every 6 hours until the untreated control cells reached 100% confluency (i.e. cell migration was complete). Data were analyzed by comparing the difference between the scratch area percentage at 0, 12 and 24 hours in Baf-A1 and CQ treated cells versus controls using Wimasis Image Analysis software (Wimasis Image Analysis, Munich, Germany).