Sodium pyruvate

Human U251-MG and U87-MG GBM cell lines were obtained from Dr. Benveniste EN (University of Alabama at Birmingham, Birmingham, AL, USA). T98G human astroglioma GBM cell line was purchased from the American Type Culture Collection (ATCC CRL-1690, Manassas, VA, USA) and CRT-MG GBM cells were established as previously described [29 (link)]. Cells were determined to be mycoplasma-free based on the Biomax Mycoplasma PCR Analysis Kit (Biomax Inc., Daejeon, Korea). U251-MG, U87-MG, and T89G-MG cell lines were grown in minimum essential medium (MEM; Welgene, Gyeongsan, Korea) supplemented with 1% of 100 mM sodium pyruvate (Welgene, Gyeongsan, Korea), 1% 100× penicillin/streptomycin (Welgene, Gyeongsan, Korea), and 10% fetal bovine serum (FBS; Welgene, Gyeongsan, Korea). CRT-MG cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Gyeongsan, Korea) supplemented with 1% 100× penicillin/streptomycin and 10% FBS. Cells were cultured in 100 mm cell culture dishes in an incubator at 37 °C and 5% CO₂ and sub-cultured every 3 days after they reached approximately 90% confluence. The cells were not cultured for more than 15 passages.

BMSCs were obtained and maintained from WT and KO mice^{56 (link)}. Briefly, bone marrow (BM) cells were isolated from the femurs and tibias of 8-week-old mice and cultured in Dulbecco’s modified Eagle medium (Welgene LM 001-05) supplemented with 10% fetal bovine serum (FBS; Welgene S 001–01), 10% horse serum (Welgene S 104–01), 2 mM L-glutamine (Welgene LS 002–01), 2 mM sodium pyruvate (Welgene LS 002–02), and 100 U ml⁻¹ penicillin/streptomycin (Welgene LS 202–02) at 37 °C and 5% CO₂. Non-adherent cells were removed by washing with PBS after overnight incubation. Adherent cells were cultured and fresh medium was added every 2 days. After 4 weeks of culture, cells were harvested by incubation with trypsin/EDTA (Welgene LS 015–01) for 2 min at 37 °C and plated onto culture dishes. At two weeks after the first passage, cells were harvested and cultured under the same conditions for subsequent passages (passages 2–7). OP9 BMSC line (ATCC CRL-2749) was maintained in alpha MEM (Welgene LM 008-02) supplemented with 20% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO₂. For induction of VCAM-1, OP9 cells were treated with TNF-α (100 ng ml⁻¹; R&D Systems 410-MT) for the indicated time period. To inhibit Ezh activity, OP9 cells were treated with various concentrations of GSK126, an Ezh2 inhibitor (Xcessbio Biosciences M60071-2), for 48 h.

Human leukemic lymphoma U937 cells were cultured on 100 mm cell culture plates in RPMI 1640 medium (Caisson Labs, Logan, UT, USA) with 10% fetal bovine serum (Thermo Scientific, Waltham, MA, USA), 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) and 1% sodium pyruvate (Welgene, Seoul, Korea) at 37°C in an incubator (Thermo Scientific) containing 95% air and 5% CO₂. The cells were differentiated by treatment with phorbol myristate acetate (PMA, 10 μg/ml, Sigma, St Louis, MO) for 72 h [1 (link)].

Every strain used in the study was purchased from the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea) or the American Type Culture Collection (ATCC, Manassas, VA, USA). The following bacterial strains were used in this study: Escherichia coli KCCM 11234 (E. coli), Pseudomonas aeruginosa ATCC 9027 (P. aeruginosa), Bacillus cereus KCCM 21366 (B. cereus), Staphylococcus aureus KCCM 11335 (S. aureus), and methicillin-resistant S. aureus ATCC 33591 (MRSA). Bacterial strains were maintained in tryptic soy broth (TSB, Difco Laboratories, Detroit, MI, USA) and cultured under shaking incubation at 37°C. In addition, the following human cell lines were used in this study: THP-1 monocytes (ATCC TIB-202), primary adipose-derived mesenchymal stem cell (hADMSC; CEFO Co., Seoul, Korea), normal bronchial epithelial cells BEAS-2B (ATCC CRL-9609), and lung carcinoma cells H460 (ATCC HTB-177). THP-1 and H460 cells were cultured in RPMI 1640 medium (Welgene Inc., Gyeongsan, Gyeongsangbuk-do, Korea) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin and streptomycin (Gibco), and sodium pyruvate (Welgene Inc.). hADMSC and BEAS-2B cells were cultured in CEFOgro™ Human MSC Growth Medium (CEFO Co.) and BEGM™ Bronchial Epithelial Cell Growth Medium BulletKit™ (Lonza, Basel, Switzerland), respectively. The cells were maintained under humidified air with 5% CO₂ at 37°C.

CAFs were isolated from lung tumors of Kras-mutant (KrasLA1) mice using magnetic-activated cell sorting with a fibroblast-specific marker, Thy1, as described previously [19 (link)]. CAFs were cultured in alpha-MEM (Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), penicillin/streptomycin (100 U/100 μg, HyClone, Logan, UT, USA), 2 mM L-glutamine (Welgene), and 1 mM sodium pyruvate (Welgene). For immortalization, CAFs were stably transfected with the TERT plasmid (pCDH-3xFLAG-TERT, a gift from Steven Artandi; Addgene 51 plasmid # 51631) using Lipofector-EXT (AptaBio, Yongin, Korea). The primary cells used in the experiments were passaged fewer than six times and were not tested for identity. Human cancer cell lines were obtained from ATCC (American Type Culture Collection; Manassas, VA, USA). 344SQ cells (a gift from Dr. Jonathan M. Kurie, University of Texas MD Anderson Cancer Center, USA) and various human cancer cell lines [A549 (lung) and HCT116 (colon)] were maintained in RPMI 1640 medium (Welgene) supplemented with 10% FBS and penicillin/streptomycin (100 U/100 μg).

The HepG2 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). HepG2 cells were cultured in a 150 mm² cell-culture dish with minimum essential medium (MEM; Welgene, Daegu, Republic of Korea) containing 10% FBS (Gibco, Grand Island, NY, USA), 100 U/mL penicillin and streptomycin (Welgene, Daegu, Republic of Korea), and 1 mM sodium pyruvate (Welgene, Daegu, Republic of Korea). All cells were incubated at 37 ℃ with 5% CO₂, and the medium was changed every other day. For the experiments, all cells were seeded at the density of 3 × 10⁴ cells/cm² and cultured in the above medium with various concentrations of B[a]P with or without quercetin or isorhamnetin for 48 h. Afterward, the cells were lysed or used for further analysis.

Benzo[a]pyrene (B[a]P), myricetin, dimethyl sulfoxide (DMSO), ammonium persulfate, nuclease P1, N,N,N’N’-Tetramethyl ethylenediamine (TEMED), protease inhibitor cocktail, phosphatase inhibitor cocktail II, phosphatase inhibitor cocktail III, glycine, sodium dodecyl sulfate (SDS), tris base, sodium chloride, tween 20, and 2-mercaptoethanol were obtained from Sigma-Aldrich Chemical (St. Louis, MO, USA). Sodium pyruvate, penicillin-streptomycin, and trypsin-ethylenediaminetetraacetic acid (EDTA) were obtained from Welgene (Daegu, Korea). Alkaline phosphatase (AP) was purchased from Takara Bio Inc (Shiga, Japan). Phosphate-buffered saline (PBS) was purchased from Biosesang (Seongnam, Korea). A total of 30% acrylamide/bis solution was purchased from Bio-Rad (Hercules, CA, USA). Antibodies, such as CYP1A1, CYP1B1, GST, ABCC2, β-actin, and HRP-conjugated anti-rabbit immunoglobulin G (IgG), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).