Bovine calf serum (bcs)

Human embryonic kidney-293 (HEK293), A549 lung carcinoma, and Vero cells that were originally derived from kidney epithelial cells extracted from an African green monkey (Chlorocebus sp.) (all cell lines were from the American Type Culture Collection (ATCC)) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% heat-inactivated bovine calf serum (GE Healthcare Life Sciences, Chicago, IL, USA). All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂ and were confirmed to be mycoplasma-free prior to use (MycoAlert PLUS detection kit, Lonza, TX, USA).

HEK-293 were cultured in DMEM high-glucose (GE Healthcare cat# SH30081.01) containing 10% bovine calf serum (GE Healthcare cat# SH30072.03), 2 mM L-glutamate (Wisent cat# 609065, 100 IU penicillin and 100 µg/ml streptomycin (Wisent cat# 450201). U2OS were cultured in DMEM high-glucose containing 10% tetracyclin-free fetal bovine serum (FBS) (Wisent cat# 081150) 2 mM L-glutamate, 100 IU penicillin and 100 g/ml streptomycin. Tetracyclin-free FBS was used to limit Cas9 expression. U2OS were starved for 2 hr in Earle’s balanced salts medium (Sigma cat# E2888).

HeLa cells and HPV16-positive SiHa cells were cultured in Dulbecco’s modified Eagle medium (GE Healthcare Life Science HyClone Laboratories) with 10% bovine calf serum (GE Healthcare Life Science HyClone Laboratories) and 1% penicillin-streptomycin (Gibco Thermo Fisher Science).

Cell lines were cultured in DMEM high-glucose (GE Healthcare cat# SH30081.01) containing 10% bovine calf serum (GE Healthcare cat# SH30072.03), 2 mM L-glutamate (Wisent cat# 609065), 100 IU penicillin and 100 µg/ml streptomycin (Wisent cat# 450201). Serum starvation media: DMEM high-glucose containing 2 mM L-glutamate, 100 IU penicillin and 100 µg/ml streptomycin. Cell lines were tested for mycoplasma contamination routinely using the mycoplasma detection kit (Lonza; cat# LT07-318). Earle′s Balanced Salts (EBSS) was purchased from Sigma (E2888).

Mouse 3T3-L1 fibroblasts were maintained in DMEM-low glucose containing 10% bovine calf serum (GE Healthcare, UK Ltd., Amersham, UK) and supplemented with 1% penicillin/streptomycin. The differentiation of mouse 3T3-L1 fibroblasts to mature adipocytes was performed by exposing post-confluent cells for 2 days to an induction medium. The induction medium consisted of DMEM-high glucose containing 10% FBS (Capricorn Scientific, Ebsdorfergrund, Germany), 1% penicillin/streptomycin, 10 µg/mL insulin, 2.5 µM dexamethasone and 0.5 mM isobutylmethylxanthine. After 2 days, the medium was changed to a maturation medium. The maturation medium consisted of DMEM-high glucose containing 10% FBS, 1% penicillin/streptomycin and 10 µg/mL insulin. The mature medium was exchanged every other day until day 12. Cells were serum-starved for 12 h prior to starting the experiment. RAW264.7 cells were obtained from the RIKEN BRC Cell Bank (Ibaraki, Japan), and were grown in DMEM-high glucose containing 10% FBS and 1% penicillin/streptomycin.

The 3T3-L1 cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (DMEM; Wako Pure Chemical Industries, Ltd) supplemented with 10% bovine calf serum (GE Healthcare) and 5% CO₂ at 37 °C. At 2 days after confluence (day 0), differentiation was induced by exchanging the previous medium for DMEM containing 10% fetal bovine serum (GE Healthcare), 1 μg/ml insulin (Wako), 0.25 μM dexamethasone (Nacalai Tesque, Inc), and 0.5 mM 3-isobutyl-1-methylxanthine (Nacalai), for 48 h. At day 2, the culture medium was changed with DMEM containing 10% fetal bovine serum and 1 μg/ml insulin. At days 4 and 6, the culture medium was changed with DMEM containing 10% fetal bovine serum (47 (link)). At day 7, we observed LDs using an inverted microscope (IX-71; Olympus Life Science).

BALB/3T3 mouse fibroblast cells, GP2-293 retroviral packaging cells, 293 T and 293 A human embryonic kidney epithelial cells, 293 A and FO myeloma cells were cultured in DMEM (Sigma-Aldrich) containing 10% heat-inactivated bovine calf serum (GE Healthcare Life Science), and 100 units/mL penicillin, and streptomycin (Thermo Fisher Scientific) at 37 °C in a humidified atmosphere containing 5% CO₂.