Gibco penicillin streptomycin

Cryopreserved human primary white SAT preadipocytes (Zen-Bio catalog # SP-F-2, lot L120116E) were seeded into PromoCell Pad growth medium (PromoCell C-27410) with 1% Gibco Penicillin-Streptomycin (ThermoFisher 15140122) and cultured according to PromoCell Pad culturing protocols. Cells were maintained in a monolayer culture at 37°C and 5% CO₂. Cells were propagated for the full experiment and not cultured beyond 5 passages.
The plating and differentiation of cells were staggered such that timepoints 1d, 2d, and 4d were collected at the same time, and timepoints 7d and 14d were collected at the same time. The 0d (ASPCs) timepoint was collected separately. To induce adipogenesis, cells were plated at confluency into 12-well plates for RNA-seq (4 isogenic biological replicates per time point) and the following day, adipogenesis was initiated using preadipocyte differentiation medium (PromoCell C-27436). The 1d and 2d timepoints were collected before any further media changes. For all other differentiation timepoints, 72 h after the preadipocyte differentiation medium was added, it was replaced with adipocyte nutrition medium (PromoCell C-27438), following PromoCell ASPC differentiation protocols.

Jurkat E6–1 T cells (ATCC TIB-152) that express CD3ɣ -GFP were cultured in RPMI 1640 Medium (Gibco, USA, CAT#: 11875119) supplemented with 10% Fetal Calf Serum (FCS) (Gibco, USA, CAT#: 14190-149) and 1% Gibco Penicillin Streptomycin (Thermo Fisher Scientific, CAT# 15140-122) in a 5% CO₂ humidified atmosphere at 37 °C. Cells were incubated on the immobilizing surface in growth medium for 30 min prior to imaging. The growth medium was replaced with pre-warmed Imaging Buffer consisting of HBSS (Life Technologies, USA, CAT#: 14175-095) supplemented with 1% FCS right before imaging. For cells imaged on the cover glass surface, cells were incubated in growth medium without FBS for 30 min at 37 °C and 5% CO₂, then growth medium was replaced with pre-warmed imaging buffer without 1% FCS. Monoclonal antibody against CD3ε (clone: OKT3, CAT#: BE0001-2-25MG) was purchased from Bio X Cell, USA.

HEK293T cells (Tiscornia et al., 2006 (link)) and COS7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 1% Gibco^™ penicillin/streptomycin (Thermo Fisher Scientific) at 37°C under 5% CO₂. For transfection, HEK293T cells were transiently transfected with the plasmid DNA using the calcium phosphate transfection method (Tiscornia et al., 2006 (link)), and cultured for 24 h before harvesting. For oxidative stress, COS7 cells were treated with 0.5 mM sodium arsenite in medium for 30 min and controls were incubated with water in medium. For proteasome inhibition, COS7 cells were treated with 10 μM sodium MG132 (Sigma-Aldrich) in medium for 3 h and controls were incubated with DMSO in medium.

Nthy-ori 3.1 (a non-transformed human follicular cell line obtained by the European Collection of Authenticated Cell Cultures) and TPC1 (a human RET/PTC rearrangement PTC cell line; from Dr. M. Takahashi, Nagoya University, Japan) were cultured in RPMI 1640 (WELGENE Inc. Republic of Korea) supplemented with 10% fetal bovine serum (HyClone, USA) and 1% Gibco penicillin/streptomycin (Thermo Fisher Scientific Inc.) at 37 °C and 5% CO₂.

Pheochromocytoma cells are derived from the rat adrenal medulla (Tharushi Perera et al., 2022 ▸ ). The PC 12 cell line used in this study was purchased from the American Type Culture Collection (ATCC, USA) and cultured in a complete Gibco RPMI medium (Thermo Fisher Scientific, Australia) supplemented with 10% Gibco horse serum (Thermo Fisher Scientific, Australia, HS), 5% Gibco foetal bovine serum (Thermo Fisher Scientific, Australia, FBS) and 1% Gibco penicillin/streptomycin (Thermo Fisher Scientific, Australia). Supplements were stored as aliquots at −20°C. Stock solutions of the PC 12 cells were prepared in a medium containing 90% FBS and 10% DMSO and stored in liquid nitro­gen. The cells were maintained at 37°C with 5% CO₂ in a 95% humidified incubator. The medium was changed every two days and passaged accordingly when the confluence reached 90%.

All cell lines were obtained from ATCC (Virginia, USA) with the exception of EO771 cells, which were obtained through a Material Transfer Agreement with Wake Forest University. Cells were cultured in Gibco DMEM (Thermo Fisher, MA, USA) supplemented with 10% heat inactivated fetal bovine serum (Atlanta Biologicals, GA, USA) and Gibco penicillin/streptomycin (Thermo Fisher) at 37 °C with 6% CO₂. Cell lines utilized include EpH4-EV (immortalized normal murine mammary epithelium), EO771 (murine luminal BC), NF639 (murine HER2/neu-overexpressing BC), HEK293 (human embryonic kidney), and C2C12 (murine myoblasts).