Penicillin streptavidin

Cell lines, obtained from ATCC (ATCC, Manassas, VA, USA), were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza) supplemented with 10% foetal bovine serum (FBS, Saint Louis, Missouri, USA), 1× L-glutamine (Lonza, Basel, Switzerland), and 1× penicillin/streptavidin (Lonza), and maintained under standard conditions. N-Acetyl cysteine (NAC) and metformin were purchased form Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Images were captured using a Leica DM IL microscope (Leica Microsystems, Wetzlar, Germany), with a 10× Plan Fluotar objective and registered using Leica Application Suite (LAS).

The breast cancer cell lines used in the present study are listed in Table 1. The cell lines were preferred based on their ER, PgR, Her-2 characteristic, in addition to their TP53 and RB1 mutational status. Except for MCF-7, being WT for TP53 as well as RB1, the other cell lines (T47D, HTB-122 and CRL2324) each harboring a TP53 mutation previously characterized as a ‘gain of function’ (GOF) mutation and with loss of the normal allele. HTB-122 in addition harbor partly deleted RB1, possibly causing a non-functional pRb protein.^34–37 Before use, the cell lines identities were confirmed by STR based DNA fingerprinting using the AmpFlSTR Profiler Plus and Cofiler kits (Applied biosystems by Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. All cell lines and medium are purchased from ATCC, and all of the different medium were supplemented with 10% FBS (Lonza, Basel, Switzerland), 1% l-Glatamine (Lonza) and 1% Penicillin Streptavidin (Lonza), and in addition McCoys were supplemented with 1 μl/ml insulin (Sigma, St Louis, MO, USA) for the T47D cells. In all the assays the cells were transfected with siRNA for 24 h, followed by treatment with dimethyl sulfoxide (DMSO, referred to as untreated cells, as negative control, Sigma) or 1 μM doxorubicin (Nycomed, Zurich, Switzerland) for 24 h, with the exception of the senescence assay (see below).

Cells were fingerprinted with AmpFISTR Profiler and Cofiler plus (Applied Biosystems by Life technologies) before use. MCF-7 (HTB-22; ATCC) breast cancer cells were cultivated in EMEM (Eagle’s minimum essential medium; ATCC), HCT116 TP53+/+ (CCL-247; ATCC) and HCT116 TP53−/− colon cancer cells were cultivated in McCoy’s medium (ATCC). The media were supplemented with 10% FBS, 2% L-Glutamine and 2% Penicillin Streptavidin (Lonza). HCT116 TP53−/− were a generous gift from Dr. F. Bunz, B. Vogelstein & K. W. Kinzler at John Hopkins University and Howard Hughes Medical Institute, MD. Prioritizing high transfection efficacy, transfection was performed using 1.85 μg/ml plasmid and 1.7 μl/ml Lipofectamin-2000 (Invitrogen). Cells were treated with dimethyl sulfoxide (DMSO) as negative control (same final concentration as for the parallel cells treated with 1 μM doxorubicin in DMSO).

The B16F10-gp75 mouse melanoma cell line was generated to stably express gp75 on the cell membrane as described in [39 (link)]. B16F10-gp75 tumor cells were cultured under humidified conditions (37 °C, 5% CO₂) in medium RPMI 1640 (Gibco, Paisley, UK) supplemented with 10% heat inactivated foetal calf serum (FCS, Lonza, Verviers, Belgium), glutamine (Glutamax, Lonza), and penicillin/streptavidin (Lonza), hereafter referred to as ‘complete medium’.
Macrophages were generated from the CD14⁺ monocytes isolated from blood as described above. Monocytes were cultured for 6 days in the presence of 50 ng/mL macrophage-colony stimulating factor (M-CSF) (eBioscience, San Diego, CA, USA) in complete medium. At day 6, cells were harvested and seeded in multi well plates for the experiment, after which they were cultured for another two days in complete medium with 50 ng/mL M-CSF.

MEFs isolated from 13.5-day embryos were cultured in a 5% CO₂ and 3% O₂ incubator, in Dulbecco’s modified Eagle’s medium GlutaMAX (Gibco), with 15% fetal bovine serum (Biowest), 100 μM 2-mercaptoethanol (Millipore), 0.01 mM Non-Essential Amino Acids, and penicillin/streptavidin (Gibco) for five or fewer passages, except for 3T3 experiments, performed in a 5% CO₂ incubator for seven passages. Cells were treated for 24 hours with 10 μM Nutlin 3a (Sigma-Aldrich) (22 (link)) or 15 μM cisplatin (Sigma-Aldrich). Primary human fibroblasts at low passage (p.2 for TINF2^K280E, p.3 for NCI-226-4 and NCI-226-8, and p.4 for TERT^P704S) were thawed and cultured in fibroblast basal medium (Lonza) with 20% fetal calf serum, l-glutamin, 10 mM Hepes, penicillin/streptavidin, and gentamicin before quantitative PCR (qPCR) analysis.