Pen str

HeLa and BV2 cells were cultured in DMEM (Gibco, Gaithersburg, MD) containing 10% FBS (Corning, Corning, New York), 1% PEN/STR (Gibco), and 4 mM L-glutamine (Gibco) in a humidified incubator with 5% CO₂ at 37°C. For Seahorse, cells were seeded at 15,000 cells/well in poly-D-lysine (PDL, Sigma, St. Louis, MO) coated 96-well microplates (Agilent Technologies, Santa Clara, CA). For ATP, AlamarBlue, and LDH, cells were seeded at 10,000 cells/well in PDL coated 96-well tissue culture plates. For LIVE/DEAD flow cytometry, cells were seeded at 150,000 cells/well in 12-well tissue culture plates. For JC-1 and TMRE flow cytometry, cells were seeded at 350,000 cells/well in 6-well tissue culture plates. HeLa cells were obtained from ATCC (Manassas, VA). BV2 cells were a kind gift from Dr. Shilpa Buch (University of Nebraska Medical Center), originally provided by Dr. Sanjay Maggirwar (George Washington University).

Rabbit L-MSCs were isolated from corneo-scleral rims of a male Chinchilla rabbit obtained during the formation of a model of limbal stem cell deficiency. The surgical procedure included careful dissection of a sample of limbal tissue with 0.5 mm depth, originating 3 mm behind the limbus and extending into corneal stromal tissue at the limbus. Animals were housed and treated according to Animal Welfare Assurance of INC RAS (IN F18-00380, 2017–2022). Limbal tissues were de-epithelialized using Dispase II (Roche, Basel, Switzerland) at a concentration of 2.4 U/mL overnight at +4 °C and next day at +37 °C for 30 min. Fragments of de-epithelized tissue were placed on culture dishes and covered with coverslips. The primary culture was incubated in DMEM/F12 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA), 1000 U/mL Pen/Str (Gibco, Waltham, MA, USA), 0.5 ng/mL amphotericin B (Gibco, Waltham, MA, USA). After reaching 80–90% confluency, cultures were harvested with 0.25% trypsin-EDTA (Gibco, Waltham, MA, USA) and cultured for 10 passages.
Rabbit BM-MSCs were kindly provided by Dr. Alexandrova S.A. from the Cell Technologies Center of the Institute of Cytology, Russian Academy of Sciences.
All cultures were maintained in DMEM/F12 medium supplemented with 10% FBS, 1000 U/mL Pen/Str in 5% CO₂ in a humidified incubator at 37 °C.

Human AML cell lines (KG1 and HL-60) and human colorectal cancer cell lines (DLD1 and HCT116) were obtained from the Korean Cell Line Bank (Seoul, South Korea). KG1, HL-60, DLD1, and HCT116 cells were cultured in RPMI-1640 Medium (Cytiva, SH30027.01, Marlborough, MA, USA), supplemented with 10% fetal bovine serum (FBS; Capricorn Scientific, FBS-12A, Ebsdorfergrund, Germany), 1% L-glutamine (Gibco, 25030-81, Waltham, MA, USA), 1% N-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES; Gibco, 15630-080) buffer, and 1% penicillin/streptomycin (PEN/STR; Gibco, 15140-122) at 37 °C in a 5% CO₂ incubator. The primary antibodies used in this study were anti-β-actin (1:5000 dilution; sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA), anti-pAKT (1:1000 dilution; 9271S; Cell Signaling Technology, Beverly, MA, USA), anti-p4EBP1 (1:5000 dilution; 9459S; Cell Signaling Technology, Beverly, MA, USA), and anti-pERK1/2 (1:1000 dilution; 9101S; Cell Signaling Technology, Beverly, MA, USA).