Mem vitamin solution

Earle’s balanced salt solution (EBSS) and L-glutamine were purchased from Himedia. Fetal bovine serum (FBS), penicillin_streptomycin, MEM NEAA, MEM amino acids, MEM vitamin solution, and Dulbecco’s modified eagle medium (DMEM) were obtained from Life Technologies, USA. Bovine serum albumin (BSA) was purchased from SRL (India). 3-MA, monodansylcadaverine (MDC), and osmiumtetraoxide (OsO₄) were obtained from Sigma-Aldrich (USA). Anti-LC3B and anti-Atg5 were purchased from Abcam (Cambridge, England). Anti-beclin-1, anti-β-actin, anti-mTOR, and anti-p-mTOR (Ser2448) antibodies were purchased from Cell Signaling Technologies. Anti-Werner antibody and horse radish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, USA. The EGFP-LC3B plasmid, which encodes a fusion protein of EGFP and LC3B, was a kind gift from Prof. Tamatsu Yoshimori (Japan). pBABE-puro mCherry-EGFP-LC3B which encodes a fusion protein EGFP, mCherry and LC3B, was a generous gift from Dr. Jayanta Debnath (Department of Pathology, University of California, San Francisco). siRNA against human WRN was purchased from Invitrogen, USA.

Three established human, non-immortalized USC cell lines (ARK1, ARK2 and SPEC2) were generously provided in 2011 by Dr. I. Fidler (MD Anderson, Houston, TX) and Dr. A. Santin (Yale University, New Haven, CT) and have been characterized in previous reports.(30 , 31 (link)). Each cell line was derived from a patient with USC. We authenticated these cell lines by confirming the HER2 protein and gene status, as well as serous histology via pathologic review. ARK1 and ARK2 cell lines were cultured in RPMI 1640 medium (Corning) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin (Life Technologies). SPEC2 cells were cultured in MEM medium containing Earle's salts and L-glutamine, supplemented with 10% FBS, 1 mM sodium pyruvate, 2% MEM Vitamin Solution and 1% MEM Non-Essential Amino Acids (Life Technologies, Grand Island, NY). BT-474 breast cancer cells were cultured in DMEM/F12 medium with L-glutamine, supplemented with 10% FBS. All cell lines were maintained in an atmosphere containing in 5% CO₂ at 37 °C.

Primary rat embryonic ventral spinal cord cultures were prepared as described previously (Loeb and Fischbach, 1997 (link)). Briefly, ventral spinal cords were dissected from embryos from timed pregnant Sprague Dawley rats (Harlan) using the ventral two-third of the spinal cords of embryonic day 15 (E15) rat embryos. Cells were plated on laminin and poly-D-lysine-coated 24-well plates at density of 200,000 cells/cm² in Leibovitz's L-15 Glutamax Medium supplemented with N-2 supplement, MEM vitamin solution, penicillin/streptomycin (Life Technologies), 6 mM NaHCO₃, 6 µg/ml chick E11 pectoral muscle extract, and 54 µg/ml imidazole. Cultures were maintained at 37°C with 5% CO₂. At day in vitro 3 (DIV3), cultures were treated with BDNF, GDNF, or NT-3 for indicated time periods. In some experiments, inhibitors were added 30 min before the neurotrophic factors. At the end of treatments, cells were washed with cold PBS and subjected to total RNA or protein extraction.

MEF cells, HEK293T cells, A549 cells, and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Cat. No. 12430) supplemented with 10% fetal bovine serum (FBS) in a 37 °C humidified incubator with 5% CO₂. Amino acid-containing (DMEMK) and -free (DMEMK-AA) media were made based on the formulation of DMEM media (Life Technologies, Cat. No.12430). Stocks for D-Glucose (100×), inorganic salts (40×), and other components (40×) were made individually. For vitamins, minimum essential medium (MEM) vitamin solution (100×, Life Technologies) was used at a 1:25 dilution (final concentration, 4×). For amino acids, MEM Amino Acids Solution (50×, Invitrogen) was used at a 1:25 dilution and supplemented with Glycine, L-Serine, and L-Glutamine. Stocks were mixed together to final concentration of 1× DMEMK or 1× DMEMK-AA. For DMEMK-AA, UltraPure distilled water (Life Technologies) was added instead of amino acids. The final pH was adjusted to 7.0–7.4. DMEMK and DMEMK-AA were used for amino acid stimulation and starvation, respectively for HEK293T and MEF cells.
For glucose starvation, the cells were cultured with D-glucose-free DMEM containing 25 mM HEPES and 10% dialyzed FBS. For rapamycin treatment, rapamycin or vehicle was added to cell culture to reach a final concentration of 100 nM.