Fetal bovine serum (fbs)

Spontaneously beating cardiac syncytia were obtained from the hearts of 1- to 2-day-old CD-1® mouse pups (Charles River Laboratories Italia, Calco, Italy), as previously described [22 (link)–24 (link)] with some modifications. Briefly, beating primary cultures of murine cardiomyocytes were prepared in vitro as follows: the hearts were quickly excised, the atria were cut off, and the ventricles were minced and digested by incubation with 100 μg/ml type II collagenase (Invitrogen, Carlsbad, CA) and with 900 μg/ml pancreatin (Sigma-Aldrich, Milan, Italy) in ADS buffer (0.1 M HEPES, 0.1 M d-glucose, 0.5 M NaCl, 0.1 M KCl, 0.1 M NaH₂PO₄•H₂O, 0.1 M MgSO₄) for 15 min at 37°C. The resulting cell suspension was preplated for 2 h at 37°C to reduce the contribution of nonmyocardial cells. The unattached, cardiomyocyte-enriched cells remaining in suspension were collected, plated onto collagen-coated 35 mm Petri dishes, and covered by DMEM containing 10% horse serum, 5% fetal bovine serum, and 1× gentamicin (Roche Molecular Biochemicals, Indianapolis, IN). About 3 × 10⁵ cardiomyocytes were cultured in each Petri dish at 37°C and 5% CO₂ to form a spontaneously beating cardiac syncytium (i.e., a cardiac cell culture made by multilayers of contracting cardiomyocytes as in our previous works [25 (link), 26 (link)]).

Bacterial strains used here included derivatives of either E. coli or S. suis 2 (Table S1). Escherichia coli Topo10 and BL21 (DE3) are applied for gene cloning, and protein expression, respectively. The growth medium for E. coli and S. suis 2 is separately Luria‐Bertani (LB) broth and Todd‐Hewitt broth (THB; Difco Laboratories, Detroit, MI). These bacteria are grown at 37°C overnight. Given the selective pressure to ensure the replication of recombinant plasmids or the maintenance of engineered strains, appropriate antibiotics (Sigma, St. Louis, MO) were supplemented as follows: 100 μg/mL of Spectinomycin for S. suis; either 100 μg/mL of Ampicillin or 50 μg/mL of Kanamycin for E. coli. The two kinds of cell lines used here corresponded to the human laryngeal epithelial cell Hep‐2 (CCTCC GDC004) and the mouse macrophagocyte Raw 264.7 (ATCC TIB‐71, Rockville, MD), respectively (Table S1) (Hu et al. 2014), and cultivated at 37°C in the presence of 5% CO₂ in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Roche, Indianapolis, IN, USA),100 μg/mL gentamycin, and 5 μg/mL penicillin G (Feng et al. 2012).

Ethanol, dimethyl sulfoxide (DMSO) penicillin-streptomycin and potassium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMEM was purchased from Gibco, USA, Fetal bovine serum from Roche, US and trypsin from Invitrogen, Grand Island, New York. Tissue culture flasks, 12 and 96-well plates were obtained from TPP (Trasadingan, Switzerland). APOPercentage™ dye was obtained from Biocolor, UK. Caspase 9 and 3/7 were purchased from Promega, Madison, WI, USA. The WST-1 tetrazolium dye was obtained from (Roche Diagnostics GmbH, Mannheim, Germany).

The isolation of mammary epithelial cells and the separation of basal and luminal cells were done as described elsewhere [61 (link),62 (link)]. Once mechanically dissociated, mammary fat pads were digested (90 min, 37°C) in CO2-independent medium (Invitrogen) containing 5% fetal bovine serum, 3 mg/ml collagenase (Roche Diagnostics) and 100 U/ml hyaluronidase (Sigma). Cells were resuspended in 0.25% trypsin-EDTA (1 min), and then in 5 mg/ml dispase (Roche Diagnostics) with 0.1 mg/ml DNase I (Sigma) (5 min). Red blood cells were lysed in NH₄Cl. Basal and luminal cells were isolated from mammary epithelial cells obtained from the inguinal glands of five 12-wk-old virgin MMTV Cre mice. Cells were stained with the following antibodies: anti-CD24-FITC (clone M1/69; BD Pharmingen), anti-CD49F-PE (clone GoH3; BD Pharmingen), anti-CD45-APC (clone 30-F11; Biolegend) and anti-CD31-APC (clone MEC13.3; Biolegend). Basal (CD24-low/α6-high) and luminal (CD24-high/α6-low) cells were purified using FACSAria III (SORP) (Becton Dickinson).