HEK293 cells (CRL-1573, A.T.C.C., Manassas, VA, USA) were grown in 25 cm2 culture flasks in MEM-Earle’s media (Sigma-Aldrich, Munich, Germany) supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich), at 37 °C in a humidified 5% CO2 atmosphere. Cell line identity was confirmed by PCR single locus technology (Promega, PowerPlex 21 PCR Kit; Eurofins Medigenomix Forensik GmbH, Ebersberg, Germany) in 2018.
For transient transfection, HEK293 cells were seeded on poly-L-lysine (Sigma–Aldrich) coated coverslips in 6-well plates, at a density of 600,000 cells per well. 24 h later, cells were transfected by adding 500 µl cell culture medium (without supplements) per well, containing 10 µl polyethylenimine solution (Sigma-Aldrich, stock concentration 1 µg/µl), 2 µg EYFP- and 2 µg ECFP-plasmid DNA. Approximately 48 h after transfection, HEK293 cells were used for confocal laser scanning microscopy.
The following plasmid combinations were used: EYFP-Cldn10b wt–ECFP-Cldn10b wt, EYFP-Cldn10b fs–ECFP-Cldn10b fs, EYFP-Cldn10b wt–ECFP-Cldn10b fs, EYFP-Cldn10b fs–ECFP- Cldn10b wt. For comparison, the frameshift mutant previously found in Saudi Arabian HELIX syndrome patients37 (link) was also included: EYFP-Cldn10b fs(SA)–ECFP-Cldn10b wt.
For transient transfection, HEK293 cells were seeded on poly-L-lysine (Sigma–Aldrich) coated coverslips in 6-well plates, at a density of 600,000 cells per well. 24 h later, cells were transfected by adding 500 µl cell culture medium (without supplements) per well, containing 10 µl polyethylenimine solution (Sigma-Aldrich, stock concentration 1 µg/µl), 2 µg EYFP- and 2 µg ECFP-plasmid DNA. Approximately 48 h after transfection, HEK293 cells were used for confocal laser scanning microscopy.
The following plasmid combinations were used: EYFP-Cldn10b wt–ECFP-Cldn10b wt, EYFP-Cldn10b fs–ECFP-Cldn10b fs, EYFP-Cldn10b wt–ECFP-Cldn10b fs, EYFP-Cldn10b fs–ECFP- Cldn10b wt. For comparison, the frameshift mutant previously found in Saudi Arabian HELIX syndrome patients37 (link) was also included: EYFP-Cldn10b fs(SA)–ECFP-Cldn10b wt.
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