Primaria 6 well plate

Primary human adult RPE (haRPE) cells were isolated from donor eyes and cultured as previously described [14 (link)]. RPE cells (250,000 cells/well, passage 2 or 3) were seeded into Primaria 6-well plates (Corning, Lowell, MA, USA) and allowed to grow for at least 1 month. For treatments, media was changed to 1% FBS. Cells were primed with 5 ng/mL Interleukin-1α (IL-1α) (R & D Systems, Inc., Minneapolis, MN, USA) and 500 ng/mL lipopolysaccharide (LPS) (Sigma, St. Louis, MO, USA) for 16 h. Cells were then treated with 5 µM rotenone (Sigma), 0.2 nM Bafilomycin A (Sigma), or 5 mM ATP (Sigma) to activate inflammasomes for 24 h.

Primary hepatocytes were prepared from male mice at 8–10 weeks of age. Mice were anesthetized with ketamine and xylazine. Livers were perfused with prewarmed liver perfusion medium (17701-038, Life Technologies) for 5 min followed by liver digest medium (17703-034, Life Technologies) for 10 min at 5 ml/min. Isolated hepatocytes (5 × 10⁵ cells/well) were transferred to Primaria 6-well plates (353846, Corning), and cultured in medium 199 (M4530, Sigma) supplemented with 10% fetal bovine serum under an atmosphere of CO₂ at 37 °C. For in vitro ketogenesis, sodium octanoate (2 mM, Sigma, C-5038) was added to culture media. The concentration of total ketone bodies in the culture media was determined 2, 24 and 48 h after the addition of sodium octanoate.

Patient-derived primary ES cells (1 × 10³) and TC-32 (positive control, 6 × 10³) cells were seeded in Primaria™ 6-well plates (Corning), allowed to adhere overnight and then treated with doxorubicin or vincristine (1–200 nM). After 72 h the media were replaced, colonies maintained for an additional 7 days, fixed and stained with crystal violet (0.25% (w/v) in methanol:ddH2O; Sigma-Aldrich) [29 (link)]. Colony numbers per well were counted using Quantity One Software (Bio-Rad).

The BALB-TTM was performed as described previously [13 (link)]. In brief, 5000 cells were seeded into Corning^® Primaria™ 6-well plates (Corning #353846, Corning, NY, USA) on Day 0 and cultivated under standard conditions. Malignant cell transformation was induced by treatment with the tumor initiator 3-methylcholanthrene (MCA, Sigma-Aldrich #213942, St. Louis, MO, USA) (0.5 µg/mL dissolved in DMSO) on Days 1–4, followed by the tumor promotor 12-tetradecanoylphorbol-13-acetate (TPA, Sigma-Aldrich #79346, St. Louis, MO, USA) (0.3 µg/mL dissolved in DMSO) on Days 8–21. Consequently, cells lost their contact inhibition and started to grow over the monolayer, and as a result, characteristic cell foci of transformed cells were formed. An additional, therapeutic treatment with 1 mM sodium butyrate (dissolved in water) (Sigma-Aldrich #8.17500, St. Louis, MO, USA) was performed on Days 32–42 (Figure 1B) or on Days 32–35 (Figure 2). On Day 42, cells were fixed with methanol and Giemsa stained for better visualization of the cell foci. For all experiments, DMSO served as a solvent control and was adapted to a constant concentration of 0.05% from Day 1. Unless stated otherwise, the assays were performed with four technical replicates in four biological experiments. The number of type-III foci was counted independently by two different persons as described elsewhere [22 (link)].