Dulbecco modified eagle medium (dmem)

HMC-3 cells (ATCC: CRL-3304), originally generated by Prof. Tardieu in 1995 via SV-40-dependent immortalization (Dello Russo et al., 2018 (link)), were kindly provided by Prof. Cordian Beyer, (Institute of Neuroanatomy RWTH Aachen University) and utilized in our previous study (Voelz et al., 2021 (link)). If not stated otherwise the HMC-3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Pan Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS, Pan Biotech, Aidenbach, Germany) and 0.5% penicillin/streptomycin [P06-07100 (P/S, Pan Biotech, Aidenbach, Germany)] for a final concentration of 50 U/ml penicillin and 50 μg/ml streptomycin. Cells were maintained in a humidified incubator at 37°C and 5% CO₂. HMC-3 cells were sub-cultured every 2 days at approximately 80% confluency by trypsinization (Trypsin/EDTA, Pan Biotech, Aidenbach, Germany). Regular PCR controls against gene markers of cell types used in our laboratory ensured the purity of our HMC-3 cells. One day prior to experiments the cells were detached by trypsinization with subsequent centrifugation [400 g, 5 min, room temperature (RT)]. The cell pellet was resuspended in DMEM, 5% FBS, 0.5% P/S (all Pan Biotech, Aidenbach, Germany) and seeded in well dishes for experiments and on coverslips for ICC.

Thermo Scientific Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific Waltham, MA) was used for quantitatively measure for cellular cytotoxicity and cytolysis. HUVECs, fibroblasts and cardio myocytes were seeded in T25 flasks, 96, 24, 12 and 6 well plates and cultivated in cell specific media (EGM (Lonza, Basel, Switzerland), DMEM (Pan Bio-Tech, Aidenbach, Germany), Myocyte basal medium (Promo Cell, Heidelberg, Germany)). 1 h after shockwave treatment LDH levels of supernatant were measured via Elisa reader, according to manufacture.

Human osteosarcoma cells (U-2 OS) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, PAN Biotech) supplemented with 10% fetal bovine serum (PAN Biotech) and incubated in a humidified chamber with 5% CO₂ at 37 °C.

Mouse embryo fibroblast cells (NIH3T3) were cultured in Dulbecco’s Modified Eagle’s Medium - DMEM (Thermo Fisher Scientific,) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin). 3T3-L1 preadipocytes were maintained until 90% confluence. Next, the medium was exchanged to differentiation medium – DMEM (PAN-Biotech GmbH, Aidenbach, DE) supplemented with 10% FBS (Thermo Fisher Scientific), 0.5 mM isobutylmethylxanthine - IBMX (Sigma-Aldrich), 1 µg/mL insulin and 1 µM dexamethasone (Sigma-Al- drich), for 3 days. Next, adipocytes were maintained for maturation until day 12 in DMEM supplemented with 10% FBS and 1 µg/mL insulin. All cell lines were cultured in 5% CO2 atmosphere at 37 °C and were seeded onto tissue culture plates one day prior to the start of the experiments.

HCT116 cells were a gift from B. Vogelstein (Baltimore, USA). H1299 cells with inducible p53^wt or p53^K120R were kindly provided by S. B. McMahon (Philadelphia, USA) [25 (link)]. RKO ATCC cells were from the DSMZ Braunschweig and a gift from M. Zörnig (Frankfurt/Main, Germany). DNA fingerprinting analysis using eight different, highly polymorphic short tandem repeat loci confirmed cell authenticity (conducted by the Leibniz Institute DSMZ, Braunschweig, Germany). Samples were tested negative for mitochondrial DNA from rodent cells (mouse, rat, hamster). Cells were maintained in high glucose (4.5 g·L⁻¹) DMEM with stable glutamine, 10% fetal calf serum (FCS), and 250 µg·mL⁻¹ gentamycin (Thermo Fisher Scientific, Waltham, MA, USA). HROC/HHC (Hansestadt Rostock/Hansestadt Hamburg colon cancer) cells were isolated from excised CRCs according to Ref. [26 ]. Low‐passaged cell lines were maintained in DMEM containing 10% FCS (Pan Biotech, Aidenbach, Germany). A. Poth (Roßdorf, Germany) kindly provided BALB/c‐3T3‐A31‐1‐1 cells from Hatano Research Institute of Japan. These were maintained in DMEM/HAM's F‐12 (3.0 g·L⁻¹ glucose; Biochrom, Berlin, Germany), 5% FCS, and 100 U·mL⁻¹ penicillin/streptomycin. Cells between passages 20–40 were used for the BALB transformation assay (˜ 70% confluence). All cells were cultivated in a humidified incubator at 37 °C with 5% CO₂.

BODIPY™ 558/568 C12 (4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid, Thermo Fischer Scientific), Hoechst (Sigma), sodium arsenite (Fischer Chemical), cycloheximide (Sigma), BODIPY™ 493/503 (ThermoFischer Scientific, D3922), Rosiglitazone (Sigma), Clofibrate (Sigma), GW501516 (Sigma), 2-bromopalmitic acid (Sigma), streptavidin-HRP (Thermo Scientific), fatty acid-free BSA (PAN Biotech), DMEM (PAN Biotech), FBS (PAN Biotech), PBS (PAN Biotech), methanol (Roth), chlorophorm (Sigma), aprotinin (Roth), leupeptin (Roth), phenylmethylsulfonyl fluoride (PMSF, Sigma), Fasnall (Sigma), and kinase screening library (10505, Cayman Chemical).

FRG1 expression vector (pCMV6.XL5.FRG1) and knockdown vector (pLKO.1. FRG1sh) along with their controls were procured from OriGene and Sigma, respectively. Plasmids were purified using Plasmid Mini Kit (Qiagen) using manufacturer’s guidelines. Human embyonic kidney (HEK293T) cells were obtained from National Center for Cell Science (NCCS), Pune, India and were grown in DMEM (PAN-Biotech) with 10% FBS (PAN-Biotech). HEK293T transfection was carried out using X-tremeGENE 9 (Roche) as per manufacturer’s protocol. Human umbilical vein endothelial cells (HUVECs) were procured from HiMedia Laboratories, Mumbai. HUVECs were maintained in HiEndoXL Endothelial Cell Growth Medium (HiMedia).