Miogel

This assay was performed to examine the modulation of OSCC cells during invasion by treatment with PL metabolites. Briefly, the membranes of 6.5 nm transwell chambers with 8 µm pores (Corning, United States) were coated with 50ul of gelatinous matrix Miogel (2.4 mg/ml) with type I collagen (0.8 mg/ml) (Corning, Cat # 354236) (Salo et al., 2015 (link))and incubated for 12–16 h. Then, the cells previously treated and non-treated with PL were plated in 200ul of DMEM/F12 medium without FBS, and subjected to the invasion assay according to Rodrigues AN et al. (2022) (link) (Rodrigues et al., 2022 (link)). The invasion profile was assessed by measuring the absorbance of the cell dye at 650 nm using an Epoch plate reader (BioTek).

To verify the potential HPSE1 modulation in the capillary structure formation, the oral cancer cells, the lines, and their respective inhibited and upregulated clones were grown in a culture medium of 10% FBS. After 70% confluence, the cells were incubated with SFB 0% for more than 24 h, and the preconditioned medium from each cell was collected. A measure of 50 µl of gelatinous matrix Miogel (2.4 mg/ml) with type I collagen (0.8 mg/ml) (Corning, Cat # 354236) (Salo et al., 2015 (link)) was polymerized in 96-well plates. HUVECs were incubated without FBS in RPMI1640 medium for 24 h. The cells were suspended in RPMI-1640 and added to wells coated with Miogel at a density of 1 × 10⁴ cell medium plus 80% the preconditioned medium and incubated at 37°C for 48 h. During the experimental period, cell morphology of capillary structures was observed and photo-documented at 12 and 24 h. The formation of the tube was examined and photographed under an inverted microscope. The quantification of antiangiogenic activity was done using Motic Image Plus 2.0 software and calculated by measuring the length of the tubule wall perimeter of endothelial cells.