H0396

At DD12, the same volume of retinal maturation medium 2 supplemented with CHIR99021 and SU5402 was added to dilute Y27632. If cells did not completely cover the surface of the culture well, this procedure was skipped. At DD13, medium was changed to retinal maturation medium 2 supplemented with CHIR99021 and SU5402 without Y27632. At DD16, medium was changed to MEM / N1 / FBS medium (MEM-alpha (M-4526, Sigma) / 1% FBS / 1% N1 supplement (N-6530, Sigma) / 2 mM L-Glutamine (G7513, Sigma) / 0.1 mM NEAA / 250 mg/L L-taurine (T8691, Sigma) / 20 μg/L hydrocortisone (H-0396, Sigma) / 0.013 μg/L triiodo-thyronine (T-5516, Sigma)) which was used for human fetal RPE culture [43 (link)]. The medium was changed three times per week.

MDA-MB-231 CTRL and MDA-MB-231 MCU-KO cell clones were previously described (Tosatto et al., 2016 (link)) and maintained in DMEM/F12 medium (Gibco 31330038) supplemented with 10% FCS, 100 U/ml penicillin and 0.1 mg/ml streptomycin. MCF10A cells were maintained in DMEM/F12 supplemented with 5% horse serum, Cholera toxin (0.1 nM, Sigma Aldrich C8052), hEGF (20 ng/ml, Peprotech AF-100-15), insulin (1X, Sigma-Aldrich I9278), hydrocortisone (500 ng/ml, Sigma-Aldrich H0396), 100 U/ml penicillin and 0.1 mg/ml streptomycin. MDA-MD-468 and BT-549 cells were maintained in DMEM medium (Sigma-Aldrich D5671) supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were grown in a humidified Heraeus incubator at 37°C, with 5% CO₂. For a better comparison, all cell types were seeded 24–48 h before the experiments in DMEM/F12 medium supplemented with 10% FCS, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Transfection was performed 24 h before the experiments by TransIT-LT1 (Mirus Bio) following manufacturer instructions. The cDNA for nuclear and mitochondrial ATeam1.03 (Imamura et al., 2009 (link)), GFP-PHD (Hirose et al., 1999 (link)), and mitochondrial 4 mt-GCamp6f (Patron et al., 2014 (link)) were previously described.

The human colonic epithelial cell lines 1CT were previously described (25 (link)). 1CT cells were cultured in complete medium, composed as follows: high‐glucose Dulbecco’s Modified Eagle (DMEM) medium/medium 199 (4530, Sigma‐Aldrich Merck, Darmstadt, Germany, at ratio 4:1), supplemented with 2% fetal bovine serum (FBS; 10270-106, Gibco ThermoFisher Scientific, Waltham, MA, USA), epidermal growth factor (EGF; 20 ng/ml, E9644, Sigma‐Aldrich Merck), hydrocortisone (1 mg/ml, H0396, Sigma‐Aldrich Merck), insulin (10 mg/ml, I9278, Sigma‐Aldrich), transferrin (2 mg/ml, T0665, Sigma‐Aldrich Merck), sodium selenite (5 nM, S5261, Sigma‐Aldrich Merck), and geneticin (G418 sulfate) sulfate (50 μg/ml, G1397, Sigma‐Aldrich Merck).
Human colonic fibroblasts (CRL-1459, ATCC, Manassas, VA, USA) were maintained in Eagle’s Minimum Essential Medium (EMEM; 30-2003, ATCC, Manassas, VA, USA) supplemented with 10% of FBS (10270-106, Gibco, ThermoFisher Scientific) and 10 μg/ml of ciprofloxacin (17850, Sigma‐Aldrich Merck).