Penicillin and streptomycin

All cell lines were cultured at 37 °C in 5% CO₂. All cell lines derived from HMLE cells were cultured in MEGM (Lonza CC-3051)/DMEM F12 (Mediatech, Wembley, WA, Australia; 10090CV) (1:1) supplemented with penicillin and streptomycin (Gibco/Life Technologies), insulin (Sigma, St. Louis, MO I9278, USA), hEGF (Sigma 9644), hydrocortisone (Sigma H0888), and bovine pituitary extract. MCF7 and MDA-MB-231 cells were cultured in DMEM/F12 (Fisher, Waltham, MA, USA; 10-090-cv) supplemented with 10% fetal bovine serum (FBS) (Sigma), and penicillin and streptomycin. SUM159 cells were cultured in Ham’s F12 (Corning 10-090-cv) medium containing 10% FBS and penicillin and streptomycin. HEK293T cell lines were cultured in DMEM (Corning, Corning, NY, USA; 10-013-CV) supplemented with 10% FBS and penicillin and streptomycin. MCF10A cells were cultured in DMEM/F12 (Corning 10-090-CV) supplemented with 5% horse serum (Sigma H1138), 20 ng/mL human epidermal growth factor (Sigma E9644), 0.5 µg/ml hydrocortisone, 100 ng/mL cholera toxin (Sigma C-8052), 5 µg/mL insulin, and penicillin and streptomycin.

HeLa-tat-III/LTR/d1EGFP cells [40 (link)] were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (Fisher Scientific), 1% penicillin and streptomycin (Sigma) and 1mg/mL G418 (Fisher Scientific). HeLa/LAV cells [41 (link),42 (link)] and pEAK Rapid cells (derived from HEK293 cells, Edge Biosystems) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (Fisher Scientific), and 1% penicillin and streptomycin (Sigma). Transient transfections of both HeLa-tat-III/LTR/d1EGFP and HeLa/LAV cells was performed using the Trans-IT HeLa-MONSTER transfection kit (Mirus). Transient transfection of pEAK Rapid cells was performed using the Trans-IT 2020 transfection kit (Mirus). Cells were harvested 48 hours post-transfection.

Total cells were extracted from femoral or tibial bone marrow from both WT and KO mice. These cells were cultivated in RPMI 1640 media (Gibco, Life Technologies) supplemented with 20% heat-inactivated fetal calf serum (Gibco, Life Technologies), 1% of penicillin and streptomycin (Sigma-Aldrich) and 30% of L-Cell Conditioned Media as a source of Macrophage Colony Stimulating Factor (M-CSF) for 7–9 days. After this period, the medium was removed and changed to RPMI 1640 media (Gibco, Life Technologies) supplemented with 20% heat-inactivated fetal calf serum (Gibco, Life Technologies), 1% of penicillin and streptomycin (Sigma-Aldrich) with specific M1 (Lipopolysaccharide [LPS] 1 μg/mL (Sigma-Aldrich) and IFN-γ 50 ng/mL; (R&D System, Minneapolis, MN) or M2 (IL-4 50 ng/mL (R&D System)or TGF-β1 50 ng/mL, (R&D System) stimuli. To identify BMDMs (bone marrow-derived macrophage) cells were incubated with Fc-block (conditioned media) followed by murine—anti-F4/80 (macrophage pan marker)—PE incubation (Caltag Laboratories South San Francisco, CA) and then visualized by flow cytometry in a FACs-Calibur (BD Biosciences—Pharmingen). These experiments showed that these adherent cells were >93% of F4/80 positive in WT and >95% of KO cells in each 10⁷ isolated cells.

Jurkat cells were purchased from ATCC and cultured in RPMI-1640 with 10% (vol/vol) fetal calf serum (Sigma, St. Louis, MO, United States; endotoxin NMT 10.0 EU/mL) supplemented with 2 mM glutamine and 100 units/mL of penicillin and streptomycin (Sigma). HEK293T cells were purchased from ATCC and cultured in Dulbecco modified eagle medium (4.5 g/L) with 10% (vol/vol) fetal calf serum and 100 units/mL of penicillin and streptomycin purchased from Sigma as described above.

The human epithelial ovarian cancer cell lines HEY and COV318 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). COV318 and HEY cells were grown in DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen, San Diego, CA), L-glutamine (0.3 g/l) and 1% (v/v) penicillin and streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a humidified incubator with 5% CO₂ atmosphere. The cisplatin-resistant ovarian cancer cell lines (PEO4 and A2780CP) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). These cells were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen, San Diego, CA), L-glutamine and 1% (v/v) penicillin and streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in adherent cultures at 37°C in a humidified incubator with 5% CO₂ atmosphere. To maintain the resistance, 1 µM cisplatin was added to the medium of A2780CP and PEO4 cells every two to three passages. All cell lines were tested for mycoplasma contaminations and STR profiled for authentication.

BRIN-BD11 rat insulin-secreting cells were maintained in Roswell Park Memorial Institute (RPMI) media (Sigma-Aldrich, CA, USA), supplemented with 10% foetal bovine serum (FBS) (Fisher Biotec, WA, Australia) and 1% penicillin and streptomycin (Sigma-Aldrich). HepG2 human hepatocellular carcinoma cells were maintained in Dulbecco’s Modified Eagle’s medium: Ham’s F-12 Nutrient Mixture (DMEM:F12 media) (Sigma-Aldrich) supplemented with 10% FBS and 1% penicillin and streptomycin. Cells were maintained in 25 cm² or 75 cm² tissue culture flasks at 37 °C in a humidified incubator equilibrated with 5% CO₂. All cells tested negative for mycoplasma contamination. Cells were seeded in 6-well plates, 96-well plates, 25 cm² vented tissue culture flasks or 75 cm² vented tissue culture flasks and allowed to recover overnight prior to treatment. All cells were treated in media supplemented with 10% LPDS, as described in Bridgeman et al. [12 (link)].

MCF-7 cell line was isolated from malignant metastatic pleural effusion of a woman having breast adenocarcinoma. It retains several ideal characteristics particular to the mammary epithelium e.g., positivity for estrogen receptors (ER) and cytokeratin while non-reactive to desmin and vimentin (mesenchymal markers). When grown in vitro, MCF7 can form domes and the epithelial like cells that grow in monolayers with doubling time of 38 h. MCF7 had a wild type p53 gene sequence (American Type Culture Collection, ATCC, Manassas, VA, USA). For this experiment, it was cultured in phenol red free DMEM supplemented with, 1mM Sodium Pyruvate, 10% char coal stripped-fetal bovine serum and 1x Penicillin and Streptomycin (Sigma-Aldrich, ST. Louis, MO, USA). hTERT-HME1 (HME1) was from Horizon Discovery (Cambridge, United Kingdom). It was derived from human normal mammary epithelial cells immortalized by stable transfection of hTERT telomerase. It was initially cultured as per the supplier conditions then maintained for the purpose of this experiment in phenol red free DMEM/F12-ham supplemented with 10% char coal stripped-fetal bovine serum and 1x Penicillin and Streptomycin (Sigma-Aldrich). All cells were maintained at 37 °C in a humidified 5% CO₂ incubator and were split before their confluency reached 90% according to supplier instructions.