Dulbecco s modified eagle medium dmem

Human umbilical vein endothelial cells (HUVEC-ECCAC obtained from Sigma Aldrich, Steinheim am Albuch, Germany) and dermal fibroblasts (BJ CRL-2522^™—purchased from ATCC, VA, USA) were cultured in RPMI and, respectively, Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal calf serum, 50 µg/mL gentamicin and 5 ng/mL amphotericin, all procured from Biochrom AG (Berlin, Germany). Cultures were fed twice weekly and incubated in standard cell culture conditions. For experiments, the concentration of 2% FCS in medium was used.

MCF-7 cells were chosen for their hormone receptor-positive attributes, reflecting the luminal breast cancer subtype. Their estrogen and progesterone receptor presence aligns with our hormone-related focus. Our selection was guided by their relevance to our objectives, well-documented status, and specific interest in MTE's impact on this hormone receptor-positive subtype. Additionally, our choice of MCF-7 cells was influenced by the existing data on green tea phenol's interaction with ERα, while information about ERβ interactions remains limited.
MCF-7 cells were cultured on an 80% monolayer in a cell culture bottle. Dulbecco's modified Eagle medium (DMEM; 3.7 g/l NaHCO3, 4.5 g/l d-glucose, 1.028 g/l stable glutamine, and sodium pyruvate; Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS; Biochrom, Berlin, Germany) was used for cultivation. The cells were incubated with atmospheric concentrations of CO₂ of 5% at 37 °C and were then trypsinized and counted for further use.

C2C12 mouse myoblasts and HEK293T cells were cultivated in Dulbecco’s modified Eagle Medium (DMEM; Biochrom AG) supplemented with 10% fetal calf serum (FCS; Biochrom AG), 2 mM L-glutamine and penicillin (100 units/ml)/streptomycin (10 µg/ml) (PAA) at 37°C and 10% or 5% CO₂, respectively. Immortalised human myoblasts were cultured in skeletal muscle growth medium (Provitro) supplemented with supplement mix (Provitro), 50 ng/ml amphotericin, 50 µg/ml gentamicin, 10% FCS, 2 mM L-glutamine and penicillin (100 units/ml)/streptomycin (10 µg/ml) at 37°C and 5% CO₂.
For transient transfection of HEK293T cells polyethylenimine (PEI; Sigma-Aldrich) was used. C2C12 cells were transfected using Lipofectamine2000 (Invitrogen) according to manufacturer’s instructions. For siRNA-mediated knockdown of human IRS4 Lipofectamine RNAiMAX (Invitrogen) was used according to manufacturer’s instructions; as control non-targeting siRNA was used (for detailed information see Supplementary Methods). In short, cells were transfected with 25 nM siRNA on two consecutive days and experiments were performed 72 h post-transfection.

Within this study we worked with several established human squamous cell carcinoma (SCC) lines of the head and neck. The FaDu_DD [37 (link)] cells originate from the hypopharynx; SAS (JCRB Cell Bank, NIBIOHN, Osaka, Japan), Cal33 (DSMZ, Braunschweig, Germany) and UT-SCC-5 cells (DSMZ) from the tongue. The metastatic cell line Detroit562 (CLS, Eppelheim, Germany) derives from pleural effusion of a primary tumor of the pharynx. The keratinocyte cell line HaCaT (DKFZ, Heidelberg, Germany) was used as normal tissue control. These established HNSCC cell lines were cultivated as monolayer with Dulbecco’s modified Eagle medium (DMEM; Biochrom, Cambridge, UK) containing 10% fetal calf serum (FCS, Sigma-Aldrich, St. Louis, MO, USA), 2% HEPES buffer (1 M, Biochrom), 1% non-essential amino acids (NEA 100×, Biochrom), 1% sodium pyruvate (Biochrom), and 1% penicillin/streptomycin (10,000 U/mL, Biochrom). The incubator was adjusted to 37 °C and 5% CO₂. Cells were passaged usually twice per week after a confluency of 70–80% was reached. Cells were only used for experiments until passage 15 and regularly tested for cell authentication and mycoplasma infection. Irradiated (IR) sublines were generated from indicated parental cell line by selection with at least 15 fractions of 4 Gy and analyzed together with age-matched controls [36 (link)].

If not stated otherwise, all chemicals were purchased from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) and fetal calf serum (FCS) were from Biochrom (Berlin, Germany). Plastic ware was from Sarstedt (Nümbrecht, Germany). All drugs tested in the present study were obtained from Elanco (St. Aubin, Switzerland) and they were all delivered as stocks of 10 g/L in DMSO. The drugs applied for experiments of Supplementary S3 Fig were purchased from Sigma, and prepared as stocks of 10 g/L as well.

IGF-1 and IGF-1 Long R3 (IGF-1 LR3) were purchased from BioVision Inc. (Milpitas, CA, USA). IGFBP-2, Dulbecco's Modified Eagle Medium (DMEM), penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Biochrom AG (Berlin, Germany). Insulin, dexamethasone, LY294002, and picropodophyllin (PPP) were supplied by Sigma-Aldrich (Darmstadt, Germany). 3-Isobutyl-1-methylxanthine (IBMX), S961, wortmannin, and Compound C were purchased from Biomol GmbH (Hamburg, Germany), Phoenix Biotech (Beijing, China), Merck Chemicals (Darmstadt, Germany), and BIOZOL Diagnostica Vertrieb (Eching, Germany), respectively. RevertAid First Strand cDNA Synthesis Kit, SYBR Green master mix, Bicinchoninic Acid (BCA) protein assay kit, and ECL reagent were supplied by Thermo Fisher Scientific (Dreieich, Germany). DNA primers were purchased from Eurogentec Deutschland GmbH (Köln, Germany). All other chemicals were supplied by Sigma-Aldrich (Darmstadt, Germany).

PC12 cells were proliferated in a complete medium containing high glucose Dulbecco’s modified Eagle medium (DMEM; Biochrom) with stable glutamine, 10% (v/v) horse serum (HS; Biochrom), 5% (v/v) heat-inactivated fetal calf serum (FCS; Biochrom), 1 mM sodium pyruvate, 100 U/ml penicillin and 0.1 mg/ml streptomycin. The cells were grown in 75-mm² tissue culture flasks coated with poly-L-lysine hydrobromide (Sigma-Aldrich), and incubated at 37 °C in a 5% CO₂ humidified atmosphere. The cell culture medium was changed every 2 days.