Culture flasks

The investigated cell lines, PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) were purchased from ATCC (Manassas, VA, USA). The cell culture medium RPMI 1640, the supplements FCS and l-glutamine, as well as PBS and trypsin/EDTA were purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Culture flasks, multi-well plates, and further cell culture plastics were purchased from Greiner Bio-One GmbH (Frickenhausen, Germany) and TPP (Trasadingen, Switzerland), respectively. Anti-proliferative and cytotoxic effects, respectively, of the compounds, were investigated by performing colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and CV (crystal violet)-based cell viability assays (Sigma-Aldrich, Taufkirchen, Germany), respectively.

The GP structural parameters—necessary for the construction of a representative 3D model and for understanding the variability of distinct structural parameters between valves—were extracted from SEM and FIB-SEM data. For this, GP diatoms (obtained from Goettingen, Germany) were cultivated in 50 ml culture flasks (Greiner Bio-One GmbH) in Wright’s cryptophyte medium [56 ] in a cultivation cabinet (Percival CFL LED) at 18 °C and a day/night light cycle of 12 h/12 h. The progress of cultivation was observed by an inverted light microscope (Zeiss, Axio Vert.A1).

Adipose samples (n = 4) were harvested according to the Coleman technique [13 (link)] during scheduled abdominal liposuctions in patients undergoing breast reconstruction. The study was approved by the local Ethical Committee on Human Study (PROT.N. 3676/CE), and participants provided written informed consent to take part in the study. An average of 600 mg of adipose samples was washed in phosphate-buffered saline (PBS; PAA Laboratories GmbH, Pasching, Austria), minced, and digested for 60 min in Dulbecco’s modified Eagle’s medium (DMEM; Euroclone Spa, Milan, Italy) containing 1.76 UI/mL collagenase solution (Roche Diagnostics GmbH, Mannheim, Germany) and 1% penicillin–streptomycin (P/S; 104 UI/mL and 10 mg/mL; PAA) at 37 °C with gentle agitation [14 (link)]. After enzyme inactivation by the addition of blocking media, the resulting cell suspension was centrifuged, filtered through a 100 μm cell strainer (BD Falcon, Durham, NC, USA), counted using 0.4% trypan blue (Biochrom AG, Berlin, Germany), and seeded in culture flasks (Greiner Bio-One GmbH, Frickenhausen, Germany) at a density of 100,000 cells/cm² in Quantum 333 culture medium (PAA) as reported [11 (link)]. Once an 80–90% confluence was reached, cells were detached with 0.05% trypsin/0.02% EDTA (Euroclone), counted, and seeded at a density of 6000 cells/cm².

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, and trypsin/EDTA were obtained from Lonza, Belgium. Sodium hydrogen carbonate was purchased from LOBA Chemie, India. Culture consumables and culture flasks were purchased from Greiner Bio-One (Germany).

Phytoplankton strains (Table 1) were cultivated in Enriched Natural Sea Water (ENSW) media, composed of Thau lagoon water (kept at obscurity for several months, filtered at 0.2 μm, and autoclaved) enriched with sodium nitrate, Ferric EDTA, monosodium phosphate, vitamins and other oligo-elements [35 (link)]. Monoclonal algal strains were grown in batch mode in Greiner bio-one GmbH culture flasks at 100 μmole photon m-2.S-1 (12:12 h light:dark) at a salinity of 35 and a temperature of 22 °C to 25 °C until DNA extraction.

The HCC827 and H1975 cell lines were purchased from ATCC. The EBC‐1 cell line was purchased from JCRB. The HCC827GR5 cell line was a kind gift of Dr. Pasi A. Jänne (Dana‐Farber Cancer Institute, Boston, MA). Cell line properties, including cMET and EGFR status, are summarized in Table 1.
Cell lines were maintained in culture flasks (Greiner Bio‐One GmbH, Frickenhausen, Germany) at 37°C in an atmosphere of 5% CO₂. The EBC‐1 cell line was cultured in DMEM supplemented with 10% fetal bovine serum (BioWest, Nuaillé, France), 100 IU/ml penicillin and 100 µg/ml streptomycin. The HCC827, HCC827GR, and H1975 were cultured in RPMI1640 with 10% fetal bovine serum, 20 mM HEPES and 100 IU/ml penicillin and 100 µg/ml streptomycin.
Crizotinib and erlotinib were purchased from SelleckChem (Houston, TX). Drugs were reconstituted in dimethyl sulfoxide (DMSO), and diluted in phosphate buffered saline (PBS) immediately before use. Bafilomycin A1 was purchased from LC‐Laboratories (Woburn, MA).