Earle s balanced salt solution ebss

For glucose starvation, INS(832/13) and islet cells were grown for 24 h in complete RPMI1640 medium containing 2.8 mmol/l glucose (low glucose, LG). Controls/non-treated (NT) INS(832/13) cells were grown in complete RPMI1640 medium containing 11.1 mmol/l glucose. Cells were also incubated with 0.5 μmol/l rapamycin, dissolved in 0.04% DMSO (an autophagy inducer; Enzo, Plymouth Meeting, PA, USA [24 h incubation]), 10 μmol/l chloroquine (a lysosomal inhibitor; Enzo [24 h incubation]) or in amino-acid- and serum-free buffer (Earle’s Balanced Salt Solution [EBSS], Sigma Aldrich [4 h incubation]).

2fTGH (human fibrosarcoma), Flag-HA-TG2 2fTGH, GFP-LC3 2fTGH and RFP-GFP-LC3 2fTGH cell lines were cultured in Dulbecco's modified Eagle's medium (Lonza) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 μg/ml streptomycin and 100 units/ml penicillin at 37°C. For generation of the TG2 stably expressing cells (Flag-HA-TG2 2fTGH), 2fTGH cells were transfected with pLPCX-TG2-HA-Flag using CaCl₂ method. Autophagy was induced by using 1 μM rapamycin (Rapam, Sigma-Aldrich) or 10 mM 2-deoxy-D-glucose (2-DG, Sigma-Aldrich). For amino acids starvation (Starv), cells were washed twice in the amino-acid-free medium Earle's Balanced Salt Solution (EBSS) (Sigma-Aldrich) and incubated in the same medium for the indicated periods. In order to inhibit autophagy, cells were incubated in full medium with 20 mM ammonium chloride (NH₄Cl, Sigma-Aldrich) for the indicated period. For proteasome inhibition, cells were incubated with 5 μM MG132 (Z-Leu-Leu-Leu-al; Sigma Aldrich) for 4 h. To induce mitochondrial damage, cells were incubated in full medium with 15 μM carbonyl cyanide m-chrolorophenyl hydrazine (CCCP, Sigma-Aldrich). TG2 transamidating activity was inhibited by incubating the cells with 40 μM Z-DON (Zedira) for 24 hours, concomitantly with starvation.

All cell lines were grown and maintained in Dulbecco’s Modified Eagle Medium (Corning) supplemented with 10% fetal bovine serum (Gibco) and 1x Penicillin-Streptomycin (Thermo Fisher Scientific). To depolarize cells for mitophagy induction, the growth medium was supplemented with 10 µM Oligomycin and 5 µM Antimycin A (Sigma-Aldrich), or with 10 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (Medchem Express). To induce starvation autophagy, cells were washed into Earle’s Balanced Salt Solution (EBSS) (Sigma-Aldrich).

Formic acid (HPLC grade) was purchased from Dikma Technologies Inc. (Lake Forest, CA, USA). Water (HPLC grade) was obtained from Fisher Scientific (Geel, Belgium). Acetonitrile (HPLC grade) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbeco’s modified Eagle’s medium (DMEM) was from Hyclone (Beijing, China). Fetal Bovin serum and GluMAX were from Life Technologies Corporation (Grand Island, NY, USA). Trypsin-EDTA (0.25%) was from Macgene (Beijing, China), and Pbs was from Solarbi science & Technology Co. Ltd. (Beijing, China). Earle’s Balanced Salt Solution (EBSS) was from Sigma-Aldrich (St. Louis, MO, USA). MTT Assay was from Solarbio life sciences (Beijing, China). Antibodies were purchased as follows: rabbit anti-LC3 (Cell Signaling Technology, 3868S), mouse anti-beta-Actin (TransGene Biotech, A005), Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, 7074S), Goat anti-mouse IgG-HRP (Abmart, M21001L). TRIzol was from life technologies (USA), PrimerScript^TM RT reagent Kit with gDNA eraser was from TaKaRa (USA). Ultra SYBR Mixture (with ROX I) was from CWBIO (Beijing, China).

Dimethyl sulfoxide (DMSO), Earle’s balanced salt solution (EBSS), hydrocortisone, insulin, minoxidil, phosphate-buffered saline (PBS), phenylmethylsulfonylfluoride (PMSF), 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and docosahexaenoic acid (DHA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). l-glutamine, antibiotic solution (Pen Strep) and Williams medium E were purchased from Gibco (Gibco Life Technologies, Grand Island, NY, USA). Dulbecco’s modification of Eagle’s medium (DMEM) and Fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). NE-PER nuclear and cytoplasmic extraction reagents were purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA). West-zol^TM Plus reagents were purchased from iNtRON (Seoul, Korea). X-ray film was purchased from Agfa-Gevaert (Mortsel, Belgium). Five percent minoxidil (MINOXIL^TM) was purchased from Hyundai Pharm. Co. Ltd (Cheonan, Korea).

Earle’s balanced salt solution (EBSS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against LC3B and p62/SQSTM1 were obtained from Sigma-Aldrich (L7543 and P0067), and β-actin antibody was purchased from Sigma-Aldrich (A5441).

C2C12 mouse muscle cell lines were purchased from American Type Culture Collection (ATCC, #CRL-1772) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Gibco), 100 IU/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂. Upon 70% confluence, C2C12 cells were harvested using 0.25% trypsin and 0.1% EDTA (Gibco) in PBS. To induced differentiation, C2C12 reached about 80% confluence, and the medium was changed to differentiation medium (DM), consisting in DMEM containing 2% fetal bovine serum, penicillin and streptomycin. The differentiation medium was switched daily until myotube were fully differentiated. For autophagy induction, myotube cells were rinsed with phosphate-buffered saline (PBS) and grown for 6 h in Earle’s Balanced Salt Solution (EBSS) (Sigma-Aldrich). To measure the insulin signaling pathway, myotube cells were incubated for 4 h in serum-free DMEM and then treated with insulin (100 ng/mL, Sigma-Aldrich). siRNA targeting selected genes or control, non-targeting siRNA were transfected into C2C12 myoblasts for 24 h using esiRNA (Sigma-Aldrich). C2C12 myoblasts were transfected using 2.5 ng of each esiRNA using RNAiMAX (Invitrogen), according to the manufacturer’s instructions.