Foetal bovine serum (fbs)

The murine mammary carcinoma cell line 4T1 was obtained from the American Type Culture Collection (Manassas, VA., USA). The prostate cell line RM11 was a gift from Associate professor Thomas S. Griffith (University of Minnesota, Minneapolis, MN., USA). This cell line was originally derived from a ras/myc reconstituted tumor in a Balb/c mouse [19 (link)]. The cells were grown in RPMI-1640 medium (HEPES solution for RM11 cells) supplemented with 10% Foetal Bovine Serum (Sigma-Aldrich, Steinheim, Germany), 100 units/ml penicillin, 100 μg/ml streptomycin, 1–2% L-glutamine (all from Sigma-Aldrich, Steinheim, Germany), with an addition of 1% sodium pyruvate for the RM11 cells. All cells were grown as single monolayers in a humidified incubator at 37°C in 5% CO₂ and they were seeded and used at log phase in all experiments. SV40 transformed wild type MEF cell line [12 (link)] was cultured in DMEM (Sigma-Aldrich, Steinheim, Germany), 10% Foetal Bovine Serum and 100 units/ml penicillin, 100 μg/ml streptomycin as previosly described.

The BV2 mouse microglia cell line and the human neuro cell line N2a were purchased from the Chinese Academy of Sciences Collection Committee Cell Bank (Shanghai, China). BV2 cells were cultured and passaged in RPMI 1640 supplemented with 10% foetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL streptomycin under an atmosphere with 5% CO₂ at 37 °C. Subsequently, BV2 cells were seeded in a 96-well plate at a density of 2 × 10⁵/mL and then treated with 10 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) for 4 h. Then, the cells were treated with 5 mM ATP (Sigma-Aldrich, St. Louis, MO, USA) for another 30 min. N2a cells were cultured in DMEM supplemented with 10% foetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL streptomycin in an incubator under an atmosphere with 5% CO₂ at 37 °C.

Crandell Rees feline kidney (CRFK) cells were grown on coverslips in 24-well cell culture plates in minimum essential medium (MEM; Gibco BRL) supplemented with 5 % (v/v) foetal bovine serum (Sigma-Aldrich) and 2 % (w/v) lactalbumine (BD Biosciences) at 37 °C and 5 % CO₂. Similarly, fIECs were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL)/Ham’s F12 Nutrient Mixture (Gibco BRL) (1 : 1, v/v) supplemented with 5 % (v/v) foetal bovine serum (Sigma-Aldrich) and 1 % (v/v) non-essential amino acids 100× (Gibco BRL). When reaching 60–80 % confluency, 24 h post-seeding, cells were transfected with 1 µg of plasmid DNA using Lipofectamine 2000 (Invitrogen). Six hours post-transfection, cells were washed and fresh medium with antibiotics (100 U ml⁻¹ penicillin, 0.1 mg ml⁻¹ streptomycin and 0.1 mg ml⁻¹gentamycin) was added.

Stably transfected Human Embryonic Kidney (HEK293T) cells, expressing 3xFlag-myc-CMV-26 vector (Sigma-Aldrich, UK) containing full-length human EPAC1, as described [39 (link),40 (link)], were grown in Dulbecco's modified Eagle's medium (DMEM), 10% (v/v) foetal bovine serum (Sigma-Aldrich, UK), 1% (v/v) of GlutaMAX supplement (Sigma-Aldrich, UK) and 1% (v/v) penicillin/streptomycin (Sigma-Aldrich, UK) and incubated at 37 °C in 5% (v/v) CO₂. Selection of stable cell lines was maintained by addition of 1 mg/ml G418 (Sigma-Aldrich, UK) to the growth medium. The human monocytic cell line THP-1 was cultured in RPMI 1640 medium (Fisher Scientific, UK) containing 10% (v/v) foetal bovine serum (Sigma-Aldrich, UK), 1% (v/v) penicillin/streptomycin (Sigma-Aldrich, UK) and 2 mM glutamine at 37 °C in 5% (v/v) CO₂. HUVECs were grown in EGM2 (PromoCell) at 37 °C and 5% (v/v) CO₂. Cells were passaged weekly to a maximum of six passages.

IMR90 cells were obtained from ATCC. IMR90 ER:RAS were generated by retroviral infection of IMR90 cells and have been described elsewhere (Banito et al., 2009; Barradas et al., 2009). IMR90 were cultured in DMEM (Gibco) supplemented with 10% foetal bovine serum (Sigma) and 1% antibiotic–antimycotic solution (Gibco). HMEC were cultured in Medium 171 (Gibco) supplemented with MEGS (Gibco), 10% foetal bovine serum (Sigma) and 1% antibiotic–antimycotic solution (Gibco). To induce OIS, IMR90 ER:RAS and HMEC ER:RAS were treated with 100 nM 4‐hydroxy‐tamoxifen (4OHT, Sigma) for 6 days. To induce chemotherapy‐induced senescence, IMR90 cells were treated with 0.5 μM Doxorubicin (Sigma) for 24 hr, or with 50 μM Etoposide (Sigma) for 48 hr (DMSO‐treatment was used as a control). To induce senescence by ionizing radiation, IMR90 were γ‐irradiated (7.5 Gy). For therapy‐induced senescence experiments (chemotherapy and irradiation), senolytic drugs were added 7 days after the induction of senescence.

All cell lines were freshly obtained from the European Collection of Authenticated Cell Cultures (ECACC) and regularly tested for mycoplasma contamination using the LookOut Mycoplasma qPCR Detection Kit (Sigma-Aldrich). T84 human colon carcinoma cells (ECACC 88021101) were cultured in Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham 1:1 (DMEM/F12) medium (Sigma-Aldrich) supplemented with 10% foetal bovine serum (Sigma-Aldrich) and 2.5 mM L-glutamine (Sigma-Aldrich) and used between passages 46-62. Mucin-producing LS174T human colon carcinoma cells (ECACC 87060401) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum, 4 mM L-glutamine and 1× non-essential amino acids (Sigma-Aldrich). For VDC experiments, 5×10⁵ T84 cells were seeded out on polyester Snapwell filter inserts (12 mm diameter, 0.4 μm pore, Corning) coated with 10 µg/cm² rat tail collagen type I (Sigma-Aldrich). For T84/LS174T co-cultures, 5.6×10⁴ LS174T cells were added (ratio 10:1). TEER was measured using an EndOhm chamber and EVOM resistance meter (World Precision Instruments) and values of >1000 Ω × cm² after 10-13 days of cell differentiation indicated establishment of T84 epithelial barrier function. Cells were grown at 37°C in a 5% CO₂ atmosphere.