Supplementmix

DPCs were placed in a 12-well microplate at a density of 50,000 cells per well and cultured in DPC growth medium containing supplement mix (PromoCell) in the presence of oleuropein or vehicle control (DMSO). Cells were fixed in 4% paraformaldehyde containing 0.1% Triton X-100 for 15 min at room temperature. After blocking with 5% BSA in phosphate-buffered saline (PBS) for 1 h at room temperature, the cells were incubated with β-catenin antibody (1:100) (Santa Cruz Biotechnology) overnight at 4°C. The cells were washed with PBS and incubated with Rhodamine Red-X goat anti-mouse antibody (Invitrogen) for 1 h at room temperature. Cells were counterstained with 4,6-diamidino-2-phenylindole (Invitrogen) and examined using a LSM510 Meta confocal microscope (Carl Zeiss, Oberkochen, Germany).

Human follicle dermal papilla cells (DPCs) were purchased from PromoCell (PromoCell, Heidelberg, Germany) and maintained in follicle DPC growth medium containing supplement mix (PromoCell) at 37°C in 5% CO₂. When the cells reached 80% confluence, they were subcultured in 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid-buffered saline solution, trypsin/ethylenediaminetetraacetic acid (EDTA) solution, and neutralizing solution (PromoCell). To investigate whether oleuropein regulation of LEF1, CycD-1, IGF-1, HGF, VEGF, and KGF occurs indirectly through newly synthesized transcription factors, the effect of the protein synthesis inhibitor, cycloheximide (CHX; Sigma-Aldrich, MO, USA) was determined. Trypsinized DPCs were allowed to attach overnight and then treated (60 min) with cycloheximide (10 μg/ml) and thereafter switched to medium with or without oleuropein (20 μM) in the presence of cycloheximide (10 μg/ml) for 24 h. Control cells were treated similarly except that no cyclohexmide was included at any time.

Two cardiac cell lines were applied for in-vitro experiments. Rat cardiomyoblasts, immature cells—H9C2 (obtained from the Lab of Department of Medical Biochemistry, Wroclaw Medical University) which were grown in Dulbecco's Modified Eagle's Medium glucose (Lonza) with the addition of 10% fetal bovine serum (FBS, Lonza), 2 mM Glutamine and 100 × penicillin/streptomycin (Sigma). Primary human cardiac myocytes (HCM, PromoCell GmbH, Germany) were isolated from the ventricles of the adult heart and grown in Myocyte Growth Medium (C-22070, PromoCell GmbH, Germany) with the addition of Supplement Mix (C-39275, PromoCell GmbH, Germany). For experiments, the cells were removed by trypsinizing (0.25% Trypsin–EDTA solution, Sigma), and washed with PBS. The cells were maintained in a humidified atmosphere at 37 °C and 5% CO₂.

A previously-described method was followed^{24 (link)}. Clonal myoblast cell lines were immortalized by transduction with human telomerase reverse transcriptase (hTERT) and cyclin-dependent kinase-4 (Cdk4) containing retroviral vectors, at the Institut de Myologie, Paris, as described previously^{42 (link)}. Immortalized myoblasts from a 25 year old donor^{24 (link),42 (link)} were cultured in skeletal cell growth medium (Cat No: C-23060; PromoCell GmbH, Heidelberg, Germany) containing supplement mix (Cat No: C-39365; PromoCell) with 20% Fetal Bovine Serum (Cat No: 10270; Gibco; ThermoFisher Scientific, Paisley, UK). Differentiation was induced at 80% confluency by washing the adherent myoblasts in medium lacking serum and then culturing in DMEM (Cat No: 31966-021; Gibco; ThermoFisher Scientific) supplemented with Insulin (1721nM), Transferrin (68.7 nM), Selenium (38.7 nM) (ITS-X; Cat No: 51500-056; Gibco; ThermoFisher Scientific) and Penicillin-Streptomycin (Cat No: DE17-603E; Lonza; Verviers, Belgium). After a further 4 days of cell culture, over 80% of the cells had fused into myotubes. Cells cultured for pull-down experiments were extracted in RIPA buffer (1% NP-40; 0.25% deoxycholic acid; 1 mM EDTA; 150 mM NaCl; 50 mM Tris-HCl, pH 7.4), in the presence of protease inhibitors (Sigma P8340 plus 1 mM PMSF), by sonication followed by centrifugation.

All cell lines were maintained at 37°C and 5% CO₂. iRECs were cultured on 0.1% gelatin-coated flasks in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, #BE12-604F/U1) supplemented with penicillin/streptomycin (Sigma-Aldrich, #P4333), L-glutamine (Thermo Fisher Scientific, #25030024), and 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific, #10270106). HK-2 cells were cultured in Renal Epithelial Cell Growth Medium (PromoCell, #C-26030) supplemented with SupplementMix (PromoCell, #C-39606) for a final concentration of fetal calf serum 0.5% (v/v), FBS 1.5% (v/v), epidermal growth factor (10 ng/mL), human recombinant insulin 5 μg/mL, epinephrine 0,5 μg/mL, hydrocortisone 36 ng/mL, human recombinant transferrin 5 g/mL, and triiodo-L-thyronine 4 pg/mL. Primary mouse proximal tubule epithelial cells were isolated from mouse renal cortices as previously described (Legouis et al., 2015 (link)) and were cultured in Basal Medium 2 phenol red-free (PromoCell, #C-22216) supplemented in the same way as HK-2 cells. OK cells were cultured in DMEM/F12 (Thermo Fisher Scientific, #21331020) supplemented with penicillin/streptomycin, glutamine, and 10% (v/v) FBS. With regard to identity, iRECs were provided directly by their creators (PMID: 27820600). The negative mycoplasma contamination status was confirmed by regular testing.

The human lung SQCC cell lines, H520 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and HCC95, H1703, and SK-MES-1 were obtained from the Korean Cell Line Bank (Seoul, Korea), and cultured in RPMI 1640 medium (H520, HCC95, H1703) and Dulbecco’s modified Eagle’s medium (SK-MES-1) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin–streptomycin (Hyclone, Logan, UT, USA). The HBEpC cells were obtained from PromoCell GmbH (Heidelberg, Germany) and cultured in an airway epithelial cell growth medium containing 2.46% SupplementMix (PromoCell GmbH). Cell culture was performed as previously described [16 (link)]. Each cell line was cultured in four biological replicates.

hDPCs were purchased from Promocell (Heidelberg, Germany) and cultured in basal medium supplemented with 4% fetal calf serum, 0.4% bovine pituitary extract, 1 ng/mL basic fibroblast growth factor, and 5 μg/mL insulin (Supplement Mix, Promocell). Cells were maintained in a humidified incubator at 37 °C, 5% CO₂. adenosine stock solution (10 mM) was prepared just before each experiment. Before adenosine (Sigma-Aldrich, MO, USA) treatment, serum limitation was conducted by replacing the medium with fresh DMEM (Gibco, Waltham, MA, USA) supplemented with 1% FBS (Gibco, Waltham, MA, USA) and 1 ng/mL bFGF (Merck, Darmstadt, Germany) and culturing for 24 h to minimize the effects of serum and growth supplements. ZM241385, PBS603, KH7, H89, LY294002, and IWR1 were purchased from Tocris Bioscience (Bristol, UK).