Hyclone

Normal human foreskin fibroblasts (VH10) donated by Leiden University were cultured in Dulbecco’s modified minimum essential medium (DMEM, Sigma-Aldrich, Schnelldorf, Germany), supplemented with 10% bovine calf serum (HyClone, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (10,000 U penicillin and 10 mg streptomycin/mL, Sigma-Aldrich). All experiments started with fibroblasts at passage 7 (P7) grown to 80% confluence before the start of each experiment. VH10 fibroblasts were passaged weekly in 75-cm² flasks at a seeding density of 3.5 × 10⁵ cells. AHH-1 lymphoblasts (ATCC, USA, cat nr. CRL-8146) were cultured in RPMI-1640 medium with 25 mM HEPES (Sigma-Aldrich), supplemented with 10% bovine calf serum (HyClone), 1% penicillin-streptomycin, 1% L-glutamine (200 mM), and 1% sodium pyruvate solution (100 mM), all from Sigma-Aldrich. AHH-1 lymphoblasts were passaged three times weekly in 75-cm² culture flasks at a seeding density of 3.0 × 10⁶ cells during weekdays and 2.0 × 10⁶ cells during weekends. All the cells were grown at 37 °C and 5% CO₂.

The immortalized human keratinocytes (HaCaT) were from Innoprot. Human The A431 epidermoid carcinoma, murine BALB/c-3T3, and SVT2 fibroblasts were from ATCC. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% foetal bovine serum (HyClone), 2 mM L-glutamine, and antibiotics, all from Sigma-Aldrich, under a 5% CO₂-humidified atmosphere at 37 °C. To test the effects of the complexes on cell viability, cells were seeded at a density of 2.5 × 10³ cells per well in 96-well plates. After 24 h, cells were incubated with increasing concentrations (from 0.1 to 50 μM) of the four complexes. After 48 h, cell viability was assessed by the MTT assay, as previously reported in [43 (link)]. Cell viability was expressed as the percentage of viable cells in the presence of the Pt complexes compared to the controls, represented by untreated cells and cells supplemented with identical volumes of DMSO. Each sample was tested in three independent analyses, each carried out in triplicate.

MDA-MB-231 and Hs 578T cells were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). The cells were passaged for a maximum of 3 to 4 months post-resuscitation and routinely tested for mycoplasma contamination. The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). All the media and their supplements were purchased from GE Healthcare HyClone or Sigma Aldrich (Saint Louis, MO, USA). All the cells were cultured up to 90–95% confluency and the media were changed every 2–3 days. The following antibodies were purchased: from Cell Signaling Technology—anti-c-Met-Tyr1234/1235 (#3077), anti-c-Met (#4560), anti-Akt-Ser473 (#4058), anti-Akt (#9272), anti-ERK1/2-Thr202/Tyr204 (#9101), anti-ERK (#4695); from Dako Agilent—anti αSMA (IR611), from Novus Biologicals—anti-vimentin (NBP1–31327), from Cell Applications—anti-ERα36 (CY-1109), from BD—anti-E-cadherin (Clone 36, 610181), from Abcam—secondary anti-rabbit IgG DyLight 488 (ab96883), secondary anti-mouse IgG DyLight 594 (ab96873), from Sigma Aldrich—β-actin (AC-15) secondary anti-rabbit HRP-conjugated (A9169), secondary anti-mouse HRP-conjugated (A9044). Cytokines were purchased from STI–VEGF (CYT-10–10), IL-8 (chm-231-a), HGF (cyt-090-a), GM-CSM (cyt-221-a), CXCL1 (100–031S), and Peprotech-CXCL5 (300–22). HGFR inhibitor-capmatinib were purchased from Selleckchem (Cat. No. S2788).

Osteolytic sarcoma cells, NCTC 2472, stably expressing GFP were cultured in NCTC135 media (Sigma, St. Louis, MO, cat# N3262) containing 10% horse serum (HyClone; Sigma), 50 mg Geneticin (Gibco; ThermoFisher, Pittsburgh, PA, cat# 10131-035), and 2.4 g sodium bicarbonate (Sigma, cat# S5761). For implantation into animals, cells were trypsinized (0.025% trypsin) off of flasks, centrifuged at 1500 RPM for 5.5 minutes, and resuspended in 1× HBSS (Gibco) at a concentration of 10 × 10⁶ cells/mL.

C57BL/6 mice were killed by cervical dislocation, ethanol 70% was sprayed for sterilization and the skin around the leg was removed. Afterward, the leg was removed and placed in DMEM (Dulbecco’s modified Eagle’s medium) on ice. In a laminar flow cabinet, the muscle was removed, the tibia and femur were separated at the knee ligament, and the proximal and distal epiphysis removed. For bone marrow derived macrophages (BMDM) culture, bone marrow was flushed out using BMDM growth media (DMEM supplemented with 10% HyClone (Thermo Fisher Scientific), 20% L929 conditioned media and 5 mM L-Glutamine (Sigma)), and the collected cells were centrifuged at 300 × G for 10 min at 15°C and resuspended in BMDM growth media. The cells were then cultured at 37°C under 5% CO₂ atmospheric conditions, with the addition of equal volume of the appropriate media after 2 days in culture and completely replenishing the media after 4 days in culture. Every experiment was conducted using cells with 7–9 days in culture.
L929 conditioned media was prepared by growing L929 cells to confluence for 2 weeks, in RPMI 1640 (Sigma) supplemented with 10% HyClone and 5 mM L-Glutamine. The culture supernatant was collected and sterilized by filtration in 0.22 μm filters (Milipore).

Mice microglial cells and BV2 were acquired from Science (Carlsbad, CA). The cells were cultured in Dulbecco's modified Eagles medium (DMEM) with 10% HyClone, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Sigma, America) with 5% CO₂ at 37°C.