Mycoplasma

Authenticated MCF7, T47D, MCF10A, HCC1500, and 293T cells were purchased from ATCC and used for experiments within the first 10 passages. Cultures were checked for Mycoplasma every 6 months (Lonza) and cells maintained at 37°C and 5% CO₂ in RPMI or DMEM + 10% FBS and 1% Pen-Strep. GDC-0941, BYL719, GDC-0032, and GDC-0068 were purchased from Selleck, and MI-136, MI-503, and MM-102 were purchased from Cayman. siRNAs (Silencer Select) were purchased from Invitrogen (Negative control #1, 4390843; siKMT2D, s528766).

4T1 cells are TNBC cell lines derived from Balb/c murine mammary tissue and were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). RPMI medium (Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT, USA), 1 mM sodium pyruvate, 2 mM L-glutamine, and 100 U/mL penicillin with 100 U/mL streptomycin (P/S; Life Technologies, Carlsbad, CA, USA) were used to cultivate the cell line, and cells were cultured in a 5% humidified CO₂ chamber at 37 °C. 4T1 cells were infected with firefly luciferase (FLuc) by the UCLA/JCCC Viral Vector Core Lab. MDA-MB-231 cells are a TNBC cell line obtained from the ATCC. Cells were grown in DMEM medium (Thermo Fisher) supplemented as above. Both cell lines were routinely tested for mycoplasma monthly (Lonza, Bend, OR, USA), and cell lines were routinely evaluated for cellular morphology and growth characteristics. All cells were used within 6 months of resuscitation.

K562 CRISPRi cell lines used in this study were maintained in RPMI 1640 (Gibco) containing L-glutamine and 25 mM HEPES, supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were stored in humidified incubators at 37°C with 5% CO₂ and routinely checked for mycoplasma (Lonza).

RKO, H508, HepG2, Huh7, HEK293T (293T), A172, U118-MG, U87, MCF-7, MDA-MB-468, and PC3 cells were obtained from ATCC. DLD1 and HCT116 cells were a generous gift from Lukas Dow. 22RV1 and was a generous gift from Dawid Nowak. 22Rv1, PC3, and H508 cells were cultured in full RPMI (Corning, Corning, NY) supplemented with 10% fetal bovine serum (FBS) (Gemini, Sacramento, CA) and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA). All of the other cells were cultured in DMEM (Corning) supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies). HepG2 cells were grown on collagen coated plates (2 ug/cm²). Cell lines were STR fingerprinted and/or bought from ATCC directly. Cells were tested for mycoplasma (Lonza, Basel, Switzerland).
Sugarless RPMI (Life Technologies) and DMEM (Life Technologies) were used in many experiments. Glucose (Millipore-Sigma, Burlington, MA) and fructose (St. Louis, MO) powders were diluted to 1 M stock in water before filtration. This stock solution was diluted into sugarless media.
To generate the semi-trained PC3 line, the parental cells were cultured in RPMI (Life Technologies) containing 1 mM Glucose, 10 mM fructose, and 5% dFBS (Life Technologies). Cells were passaged approximately once per week. After >20 passages, semi-trained cells were cultured in a 10-mM fructose in order to generate trained PC3 cells.

The murine astrocyte type I clone (C8-D1A, CRL-2541; [60 (link)]), baby hamster kidney 21 (BHK 21), and African green monkey epithelial (Vero) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Murine neuronal NSC-34 cells were a gift from Neil Cashman, University of British Columbia, Canada [61 (link)]. All cell lines were documented to be free of mycoplasma (Lonza Bioscience, Walkersville, MD, USA) and cultured at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Flower Branch, GA, USA), streptomycin (100 μg/mL; Gibco, Grand Island, NY, USA), penicillin (100 U/mL; Gibco, Grand Island, NY, USA), and L-glutamine (2 mM; Gibco, Grand Island, NY, USA).