Ham s f 12 nutrient mixture

ARPE-19 cells are immortalised RPE cells established from the ocular globes of a 19-year old male donor as previously described^{50 (link)}, were obtained from ATCC, USA. Cells were grown in a 1:1 mixture of Dulbeco Modified Eagles Medium and Ham’s F12 nutrient mixture (Sigma Aldrich, Dorset, UK) supplemented with 10% heat inactivated foetal bovine serum (FBS) (Sigma Aldrich, Dorset, UK), at 37˚C and 5% CO₂ and passaged using 1× Trypsin–EDTA (Sigma Aldrich, Dorset, UK) at a 1:3 ratio. Cells were counted using cell dual-chamber cell counting slides (BioRad, USA) on a Bio-Rad TC10 automated cell counter (BioRad, USA), and seeded into 6-well cell culture plates (6-well plate) (Corning Incorporated, Maine, USA) at a seeding density of 2.5 × 10⁵ cells per well.

Skin biopsies were minced into 1 cm² pieces and digested with 0.5% (w/v) collagenase type V (Merck, Darmstadt, Germany) and 0.25 mg/ml thermolysin (Sigma-Aldrich, Taufkirchen, Germany) in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Darmstadt, Germany) for 3–5 hours (h) at 37°C in 5% CO₂. After enzymatic treatment, intact sweat glands were released from the digested skin and transferred with a capillary micropipette to cell culture flasks or to Nunc Lab Tek II chamber slides (#154917, Sigma-Aldrich), which were all coated with Collagen I (BD Biosciences, Heidelberg, Germany). Sweat glands were cultivated in DMEM media supplemented with 30% Ham’s F12 Nutrient Mixture (Sigma-Aldrich), 10% fetal clone serum II (Invitrogen, Carlsbad, CA, USA), 10 ng/ml epidermal growth factor (EGF, Sigma-Aldrich), 100 UI/ml penicillin (Sigma-Aldrich) and 25 μg/ml gentamicin (Sigma-Aldrich) at 37°C in a humidified atmosphere of 5% CO₂. EGF is notably required for the in vitro growth of sweat gland cells like myoepithelial cells as shown in previous studies [31 (link), 32 (link)]. The media was changed every 2–3 days and the outgrowing cells were harvested and subcultivated for following experiments.

The human cancer cell lines used for the evaluation of the Pt compounds cytotoxicity (see Table 1) were cultured in different culture media, as reported below:

Caco-2: DMEM (low glucose) medium (Sigma-Aldrich, St. Louis, MO, USA), 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), glutamine 2 mM, penicillin (100 U/mL), streptomycin (100 mg/mL), 1% non-essential amino acids.

MG-63 and SK-OV-3: DMEM (high glucose) medium (EuroClone, Pero, MI, Italy), 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), glutamine 2 mM, penicillin (100 U/mL), streptomycin (100 mg/mL).

HeLa, MCF-7 and ZL-55: RPMI 1640 (EuroClone, Pero, MI, Italy), 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), glutamine 2 mM, penicillin (100 U/mL), streptomycin (100 mg/mL).

Hep-G2: DMEM (low glucose) medium (Sigma-Aldrich, St. Louis, MO, USA), 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), glutamine 2 mM, penicillin (100 U/mL), streptomycin (100 mg/mL).

SH-SY5Y: 1:1 mixture of DMEM (high glucose) and Ham’s F-12 Nutrient Mixture (Sigma-Aldrich, St. Louis, MO, USA), 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), glutamine 2 mM, penicillin (100 U/mL), streptomycin (100 mg/mL).

All experiments were performed using Chinese Hamster ovary cells, CHO-K1 (Cat. No 85051005; European Collection of Cell Cultures, Salisbury, UK), which do not express endogenous EGFR. Cells were cultured in Ham's F-12 Nutrient Mixture (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA) and 50 μg/ml gentamycin (Life Technologies) at 37°C under 5% CO₂ atmosphere. Two days prior to imaging, cells were seeded on a glass bottom 8-well Lab-Tek chamber slide (Thermo Scientific, Rockford, IL) and transfected with one or combination of following plasmids: eGFP/pcDNA3, Lyn-eGFP/pcDNA3, Lyn-mCherry/pcDNA3, wild type or mutant eGFP-EGFR/pcDNA3, mCherry-HA-PDK1/pcDNA3, PLCγ1-mCherry/pcDNA3, pEKAREV [46 (link)] (~0.2 μg of each plasmid per well) using FuGENE6 transfection reagent (Promega, Madison, WI) according to the manufacturer’s protocol. The EGFR expression level was evaluated by the confocal microscopy. Before imaging, cells were incubated in serum-free F-12 for ≥ 20 min for serum starvation. For PKC activation experiments, cells were incubated for ≥ 20 min in serum-free F-12 containing of 200 nM phorbol 12-myristate 13-acetate (PMA; InvivoGen, Toulouse, France) and 0.02% Kolliphor EL (Sigma-Aldrich). For PKD inhibition experiments, cells were incubated with 10 μM CID755673 (Sigma-Aldrich) in the presence of 0.02% Kolliphor EL.