S11150

Human SKOV3.ip1 ovarian cancer cell lines were cultured and maintained at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM) (10-013-CV, Corning Cellgro) supplemented with 10% (v/v) fetal bovine serum (FBS) (S11150, Atlanta Biologicals, Lawrenceville, GA, USA) and 1% penicillin-streptomycin (Pen Strep) (v/v) (15240, Invitrogen). Similarly, the murine ID8 ovarian cancer cell line was cultured in DMEM supplemented with 4% FBS and 1% Penicillin Strep and 1% Insulin-Transferrin-Selenium (25-800-CR, Corning). RAW 264.7 cells and human peripheral blood mononuclear cells (hPBMCs) were cultured in 10% RPMI and 10% FBS and 1% penicillin-streptomycin]. ID8 or SKOV3ip.1 ovarian cancer cells that stably express mRuby (ID8-mRuby/SKOV3ip.1-mRuby) were constructed by lentiviral delivery of pLVX-mRuby expression vector (Stanford genomics facility). The mRuby tagged expression vector along with the packaging vector master mix PMDL(PVSV-G) and PSPAX2 lentiviral expression plasmid was transfected into HEK293T cells to generate the viral conditioned medium. Fluorescence-activated cell sorting using a BD FACS Aria II system (BD Biosciences, San Jose, CA) at the Stanford Flow Cytometry Core Facility was used to select for high mRuby-expressing cells. All cells were maintained under standard tissue culture conditions (i.e., in a humidified incubator at 37 °C supplemented with 5% CO₂).

Day 10 NCCs were washed with 1× PBS and dissociated in accutase. The cell/accutase mixture was washed by mixing the solution with FACS buffer that contains 1× DMEM (Life Technologies, 10829-018), 2% FBS (Atlanta Biologicals, S11150) and 200 mM L-Glutamine. The cells were spun twice at 200 × g for 4 min. Count the cells and incubate 1 × 10⁶ cells with mouse anti-CD49d-PECy7 (Biolegend, 304313, 1:20 in 100 μl) for 20 min on ice. After incubation, wash cells by mixing the mixture with FACS buffer and spinning twice at 200 × g for 4 min. Cells in FACS buffer were kept on ice and analyzed using Beckman Coulter CytoFLEX.

HL-1 cardiac muscle cells were purchased from MilliporeSigma. THP-1 human monocyte cells were obtained from ATCC. THP-1 cells were differentiated into macrophages by 100 ng/mL PMA for 2 days in RPMI 1640 medium. Then, THP-1 cells were cultured in hyperglycemic (25 mM) DMEM with 10% FBS during the treatment.
Frozen human peripheral blood CD14⁺ monocytes were purchased from Physicians Plasma Alliance (PPA). Monocytes were differentiated into macrophages in the presence of 50 ng/mL M-CSF (216-MC-025, R&D Systems) for 6 days in DMEM medium (catalog 11995073, Thermo Fisher Scientific) supplemented with 10% FBS (catalog S11150, Atlanta Biologicals) and 1% penicillin-streptomycin (catalog 15-140-122, Fisher Scientific). Then, the monocyte-derived macrophages were cultured in hyperglycemic (25 mM) DMEM with 10% FBS and 1% penicillin-streptomycin during treatment.

Human breast cancer cell lines MCF7 (HTB-22, ATCC) and MDA-MB-231 (HTB-26, ATCC) were maintained in α-minimum essential medium (α-MEM; SH30265, GE Healthcare), supplemented with 10% fetal bovine serum (FBS; S11150, Atlanta Biologicals) and 1% penicillin/streptomycin (P/S; 15140, GIBCO). The PC3 human prostate cancer cell line was maintained in RPMI–1640 medium (23400–021, GIBCO) supplemented with 10% FBS and 1% P/S. Head and neck squamous cell carcinoma (HNSCC) OSC19 and HN5 cells were maintained in Dulbecco’s modified Eagle medium (DMEM; SH30022FS, GE Healthcare), supplemented with 10% FBS, 1% P/S, 1 mM sodium pyruvate (11360070, GIBCO), 2 mM L-glutamine (25030081, GIBCO), 1% MEM vitamin (11120052, GIBCO), and 1% MEM non-essential amino acids (NEAA; 11140050, GIBCO). The HCT116 human colorectal carcinoma cell line and its derived cells – DNA-PKcs knock-out (KO) cells⁴¹ – were maintained in α-MEM supplemented with 10% FBS and 1% P/S. All cells were cultured in a humidified 5% CO₂ incubator at 37 °C.

Primary human gingival fibroblasts (HGF-1, CRL-2014, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture DMEM/F-12 (HyClone^TM, GE Health Life Sciences, South Logan, UT, USA) with 10% fetal bovine serum (S11150, Atlanta Biologicals, Flowery Branch, GA, USA) 100 IU Penicillin (Corning, Inc., Corning, NY, USA), 100 μg/mL Streptomycin (Corning, Inc., Corning, NY, USA) and 2 mM L-glutamine (GlutaMAX^TM, Life Technologies, Carlsbad, CA, USA) All cells used in the experiments were at passage 7. Replicates (n = 3) were seeded at a concentration of 50,000 cells/well and incubated for 3 days at 37 °C and 5% CO₂. Cells were fixed with 3.7% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 10 min at room temperature, washed with phosphate buffered saline (PBS) 3 times, and stained with Actin green and 4′,6-diamidino-2-phenylindole (DAPI) per manufacture instructions (ActinGreen^TM 488 ReadyProbes^TM, Invitrogen, Carlsbad, CA, USA; NucBlue^TM Fixed Cell Stain ReadyProbes^TM, Molecular Probes, Eugene, OR, USA). Templates were stored in 96 well plates immersed in PBS at 4 °C and were imaged within 72 h.