Histidinol

HCT116 cells were maintained in McCoy’s 5A media (Cytiva, SH30200FS), and T98G, Hela, mouse embryonic fibroblasts and 293T cells were maintained in Dulbecco’s Modified Eagle’s medium (Sigma-Aldrich, D5796) at 37°C with 5% CO₂. All media were supplemented with 10% fetal bovine serum and 1X penicillin/streptomycin (Gibco, 15070063). For experiments involving thapsigargin (Sigma, T9033), tunicamycin (Sigma, T7765) and histidinol (Sigma, H6647), media were supplemented with 1X GlutaMAX (Gibco, 35050079) and 1X MEM Non-Essential Amino Acids (Gibco, 11140050).

Sept7 floxed mouse embryonic fibroblasts were generated from E15 day embryos and maintained under standard conditions. Sept7 floxed adult tail fibroblasts (TFs) were isolated from 6–8 weeks old mice tail tips. Minced tail tips were sequentially digested with collagenase and trypsin at 37°C and plated on collagen coated dishes in DMEM supplemented with 20% serum, non-essential amino acids and antibiotics. The cells were splitted 1∶4 and maintained in the same growth medium without coated dishes. To immortalize primary TFs, cells were co-transfected with pSV40Tag encoding simian virus 40 large T antigen and pREP8 plasmid (Invitrogen) in a 10∶1 mixture; colonies were selected with 2 mM histidinol (Sigma). Jurkat cells were maintained in RPMI-1640 medium supplemented with 15% serum, 1 mM pyruvate and antibiotics. Post electroporation cells were additionally supported by 2 ng/ml IL2.

All cell lines were maintained in standard growth medium -DMEM/high glucose, 1 mM glutamine, 10% FCS (20% for MEFs), 1x Penicillin/Streptomycin. Transient transfections were performed using polyethyleneimine (PEI) reagent as described previously24 (link). Sept7 floxed mouse (Sept7^tm1Mgl)14 (link) embryonic fibroblasts were generated from E14 day embryos. Aseptically minced embryos after removing head and red organs were further disaggregated by mincing in a solution of Trypsin-EDTA/DNAseI and incubation with 5 mm glass beads with vigorous shaking at RT for 15 min. The isolated cells were transferred to a fresh tube and resuspended in standard growth medium with 50 μg/ml gentamycin, plated in T25 flasks and propagated at 37 °C with 5% CO₂. The cells were splitted 1:4 and maintained in the same growth medium. To immortalize primary MEFs, cells were co-transfected with pSV40Tag encoding simian virus 40 large T-antigen and pREP8 plasmid (Invitrogen) in a 10:1 mixture; colonies were selected with 2 mM histidinol (Sigma). For proliferation assays, MEF clones were seeded in 96 well plates and on alternate days viable cell population were quantified by WST-1 assay (Roche).