Dmem ham s f12 1 1 medium

Lox-IMVI, Malme-3M, Sk-Mel-5, and Sk-Mel-28 were obtained from the Department of Developmental Therapeutics, National Cancer Institute (NCI). WM-115, WM-266-4 were obtained from the European Collection of Authenticated Cell Cultures (ECACC) and the SK-Mel-2 cell line was obtained from the American Type Culture Collection (ATCC). Cell lines were maintained at 37 °C with 5% CO2 in RPMI-1640 medium (Sigma) with 10% FCS (BioWhittaker). DLKP-A is an adriamycin-resistant, P-gp over-expressing variant of DLKP, established in the National Institute for Cellular Biotechnology from a squamous cell lung carcinoma sample [28 (link)]. DLKP-Mitox is a mitoxantrone-resistant sub-variant of DLKP that over-expresses BCRP that was used as a positive control for BCRP expression also established in the National Institute for Cellular Biotechnology [29 (link)]. DLKP-A, DLKP-Mitox and DLKP cells were maintained in DMEM/Ham’s F12 1:1 medium (Sigma) with 5% FCS (BioWhittaker). Stock solutions of ABT-751 (10 mM) (Abbott), and elacridar (3.56 mM) (Sigma-Aldrich) were prepared in dimethyl sulfoxide (Sigma-Aldrich). Clinical formulations of docetaxel (11.6 mM) and paclitaxel (7.03 mM) were obtained from St. Vincent’s University Hospital.

Human embryonic kidney 293T (HEK293T) cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM; Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (all Thermo Fisher Scientific). For murine inner medullary collecting duct 3 (mIMCD3) cells, DMEM/Ham's F-12 (1:1) medium (Merck) was used. Cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. For transient transfection of HEK293T cells, Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) was used following standard protocols.

To investigate the consequences of a knockout of Dact1 on branching morphogenesis, a tubulomorphogenesis assay was performed using mIMCD3 cells, as previously described (De Tomasi et al. 2017 (link)). In brief, mIMCD3 cells were cultured in a three-dimensional (3-D) gel of collagen type I from rat tail (Corning, Corning, NY, USA) in 12-well plates. For each experiment, each cell clone was plated in duplicate. A thin collagen layer without cells was applied to the well, followed by a collagen layer containing 100,000 cells/ml. After the gel had solidified, 500 µl of DMEM/Ham's F-12 (1:1) medium (Merck) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (all purchased from Thermo Fisher Scientific) were added to the well. After seven days of cultivation, cells were documented using an inverted microscope (DM IL LED Fluo, Leica Microsystems) equipped with an EC3 camera (Leica Microsystems). For better visualization, 3-D cultures were fixed in 4% PFA in PBS and stained with Alexa Fluor 488 Phalloidin (#A12379; Invitrogen; dilution 1:200 in PBST). For quantification, images of 3-D cultures were blinded, and at least 60 cellular structures were classified as tubular or spherical for each cell clone in each experiment (n = 3).