24 well tissue culture dishes

Subconfluent HEK293 cells in 24-well tissue culture dishes (Becton Dickinson) were transiently transfected using Metafectene Pro (Biontex) according to the manufacturer's protocol. All wells were cotransfected with 60 ng of luciferase reporter vectors, 100 ng or as indicated in the figures of SPBP expression vector (pDEST-HA-SPBP), 100 ng of CBP expression vector (pHA-CBP), 50 ng or as indicated in the figure of pDEST-HA-NRF2 and 5 ng of a β-galactosidase expression vector. pcDNA₃HA (Invitrogen) was used to equalize the concentration of DNA in each transfection. Extracts were prepared 20 hours post transfection and analyzed essentially as described previously [27] (link). All assays were performed in triplicates and repeated at least three times.

M0505 and STOSE cell proliferation was assessed from 1 to 3 days after seeding 2 × 10⁴ cells in 24-well tissue culture dishes (Becton Dickinson) in MOSE medium. The number of viable cells was determined using the Vi-CELL XR cell viability analyzer (Beckman Coulter).

Titanium discs were placed in 24-well tissue culture dishes (Becton Dickinson), seeded with either keratinocytes or gingival fibroblasts (1 × 10⁵ cells in 1 ml DF-K medium or DMEM supplemented with penicillin (50 units/ml), streptomycin (50 μg/ml) and 5% fetal calf serum, respectively) and incubated in 5% CO₂ in air at 37°C for 24 hours. This time point was chosen since a sufficient number of cells had adhered in order for the results to be reliable but little cell division had occurred. The discs were gently washed with PBS to remove non-attached cells and stained using Live/Dead BacLight staining kit (Molecular Probes). Adherent cells were visualized using a fluorescence microscope. To evaluate how well the cells were attached to the substratum, a standardised washing procedure was used to remove loosely adhered cells [23 (link)]. Discs in culture dishes containing 1 ml PBS were shaken at 100 rpm for 2 × 5 minutes (IKA Vibrax rotary shaker, GMBH & Co., Germany). Cells remaining after this procedure were counted manually after staining with Live/Dead BacLight staining kit and image capture using fluorescence microscopy. The number of cells remaining after the wash was expressed as a ratio of control (number of cells present before washing).