Multi well cell culture plates

LLC-PK1 cells were plated into multi-well cell-culture plates (Falcon) and co-transfected with constructs indicated in the figures. After 24 h, cells were fixed with 4% paraformaldehyde and nuclei were stained with DAPI (Lonza). A total of 25 images per well were acquired using the ImageXpress Micro system (Molecular Devices) and analysed with the inbuilt Multi Wavelength Translocation analysis module. Nuclear-to-cytoplasmic ratios were calculated and the median of each distribution was reported.

HUVECs (1 × 10⁴ cells/well) in endothelial cell basal medium supplemented with growth medium supplement mix and penicillin-streptomycin were seeded on 24-well clear flat-bottom TC-treated Multiwell Cell Culture Plates (353047, Falcon, Glendale, AZ). After 1 h of OGD, the medium was replaced with IM, NSC-CM, or NSC-CM with AG490 (50 μM). After incubation at 37° C for 24 h, cells that did not migrate through the membrane were removed. The cells migrated were fixed with 4% PFA for 10 min and then stained with 0.2% crystal violet for 30 min. The stained cells were counted in three randomly selected areas using ImageJ software.

The primary human oral keratinocytes (HOKs) isolated form normal oral mucosa (ScienCell, Uppsala, Sweden) were cultured in complete serum-free oral keratinocyte medium (ScienCell, Uppsala, Sweden). The highly invasive human tongue squamous cell carcinoma HSC-3 cell line (JCRB Cell Bank; Osaka National Institute of Health Sciences, Osaka, Japan) and the tongue squamous cell carcinoma SCC-25 cell line (ATCC, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle medium with Nutrient Mixture F-12 Medium, 10% fetal bovine serum, penicillin–streptomycin, and 0.1% hydrocortisone. Cells were subcultured when they were over 90% confluent, then cells were counted and plated in multi-well cell culture plates (BD Falcon, Lawrence, KS, USA).