Dulbecco s modified eagle s medium dmem

Two cell lines were prepared with high and low GGT expression. The high GGT expression cell line was A549, established from human lung adenocarcinoma and reported as a positive control for gGlu-HMRG [9 (link)], and the low GGT expression cell lone was SW782, a noncancerous line of human pre-adipocytes.
HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Wako).
Cell blocks were constructed as follows. HepG2 cells were cultured in 100-mm plates (Nacalai Tesque) and harvested using a scraper (Nacalai Tesque). Cells in 200 μL of saline were then centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and the cells were fixed in formaldehyde saline overnight. Fixed cells were embedded in agarose gel. The cell blocks were cut into thin sections for HE and immunohistochemical staining.

For myogenic differentiation, we modified a previously reported protocol (Fig. 2a). Briefly, iPSC^MyoD were trypsinized, dissociated to single cells, and seeded on Matrigel (BD Biosciences, San Diego, CA, USA)-coated plates at a density from 4 × 10³ to 5 × 10⁴ cells/cm² (cell line-specific) in 20% (v/v) knockout serum replacement (KSR) human iPSC medium with 10 µM Y-27632. After 24 h, Dox (LKT Laboratories, St. Paul, MN, USA) was added at 1 µg/mL. On day 2, the medium was replaced with high glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) containing 5% (v/v) KSR (Thermo Fisher Scientific), 1 µg/mL Dox, 1 mM L-glutamine (Thermo Fisher Scientific), and 0.1 mM 2-mercaptoethanol (2-ME) (Thermo Fisher Scientific). The medium was changed daily. For the transient glucose deprivation experiment, the medium was replaced with glucose-free DMEM/Ham’s F-12 (Nacalai Tesque) containing 1 µg/mL Dox, 1 mM L-glutamine and 0.1 mM 2-ME for 24 h prior to the glycogen analysis. For the rhGAA rescue experiment, 1 µM Myozyme (rhGAA) (Sanofi Genzyme, Cambridge, MA, USA) was added to the medium for the last 3 days unless otherwise specified.

We used a hiPSC line; 201B6[4 (link)] for generating cardiovascular cell populations. The methods for culturing and passaging human iPSCs have been previously reported in detail [8 (link)]. Briefly, iPSCs were detached with Versene (0.48 mM EDTA solution; Life Technologies, Carlsbad, CA, USA) and plated onto Matrigel (growth factor reduced, 1:60 dilution; Life Technologies)-coated plates at a density of approximately 100,000 cells/cm² in mouse embryonic fibroblast conditioned medium [MEF-CM; Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% non-essential amino acids (NEAA) (Life Technologies)] with 4 ng/ml bFGF (Wako Pure Chemicals Industries, Osaka, Japan) for 2 days before induction. Cells were covered with Matrigel (1:60 dilution) 1 day before induction. MEF-CM was replaced with RPMI+B27 medium (RPMI1640, 2 mM L-glutamine, B27 supplement without insulin; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 ng/ml of Activin A (R&D, Minneapolis, MN, USA) for 1 day, followed by 10 ng/ml BMP4 (R&D) and 10 ng/ml bFGF for 3 days without culture medium change. At 5 days of differentiation, the culture medium was replaced with RPMI+B27 medium with 50 ng/ml VEGF₁₆₅ (Wako Pure Chemicals Industries), and culture medium was refreshed every other day.

Two milliliters of sterilized 4.05% thioglycollate medium Brewer modified (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was administered intraperitoneally into the mice, and the mice were housed for four days [14 (link),51 (link)]. After the mice were euthanized by cervical dislocation, peritoneal exudate cells were harvested by sterile lavage of the peritoneal cavity with ice-cold Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Kyoto, Japan). The cells were washed once with ice-cold DMEM, resuspended in DMEM supplemented with 10% heat-inactivated fetal bovine serum (BioWest, Nuaillé, France), 100 units/mL penicillin (Nacalai Tesque), and 100 μg/mL streptomycin (Nacalai Tesque), and then cultured at 37 °C in a humidified incubator containing 5% CO₂ for 1 h. After the nonadherent cells were removed, peritoneal exudate macrophages were used in the experiments.

HeLa (#CCL-2.2, ATCC), FLAG-RIG-I/HeLa (derived from HeLa; #CCL-2.2, ATCC), FLAG-IPS-1/HeLa (derived from HeLa; #CCL-2.2, ATCC) [33 (link)], EGFP-G3BP1/HeLa (derived from HeLa; #CCL-2.2, ATCC) [35 (link)], HEp-2 (#CCL-23, ATCC), BHK21 (#CCL-10, ATCC) cells, and MEFs (isolated from embryos under C57BL/6 background, Japan SLC, Inc.) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Nacalai Tesque) supplemented with 10% Fetal Bovine Serum (FBS) (BioWest) and 1% Penicillin-Streptomycin Mixed Solution (100 U/ml and 100 μg/ml respectively) (Nacalai Tesque).

The human breast cancer cell line MCF-7 and osteosarcoma cell line U2OS were obtained from the American Tissue Culture Collection (Manassas, VA, USA). The human breast adenocarcinoma cell line MDA-MB-231 and colon cancer cell line HCT116 were purchased from JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank) (Osaka, Japan). The human breast carcinoma T47D cells and murine embryonic fibroblasts (MEF) were purchased from Cell Biolabs, Inc. (San Diego, CA, USA) and LONZA (Walkersville, MD, USA), respectively. MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Tokyo, Japan) supplemented with 5% fetal bovine serum (FBS) (BioWest, Nuaillé, France), 100 units/mL penicillin (Nacalai Tesque), and 100 μg/mL streptomycin (Nacalai Tesque). Other cells were cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin.