High glucose dmem

As previously mentioned, we also isolated chicken primary myoblasts from the leg muscle of 11-day-old AA broiler embryos [31 (link)]. The cells were grown in high-glucose DMEM (Biological Industries, Beit Haemek, Israel) containing 15% FBS and 1% penicillin/streptomycin (Solarbio, Beijing, China) until the second generation of cells had reached >90% confluence and then changed to differentiation medium (DMEM containing 2% horse serum) for further culture. An inverted microscope was used to observe the cell status. Cells were maintained at 37 °C in a 5% CO₂ environment, and cell samples were collected at 1, 2, 3, 4, 5, 6, and 7 days of differentiation induction.

The colorectal adenocarcinoma cell lines WiDr and C₂BBe₁ were obtained from BCRC (Bioresource Collection and Research Center, Food Industry Research and Development Institute, Taiwan). STR profile performed from BCRC. The human normal colon epithelial cell line CCD841 CoN was obtained from American Type Culture Collection (ATCC). STR profile performed from ATCC The MSCs were obtained as a gift from Dr. Chao-Ling Yao (National Cheng Kung University).
The WiDr cells were cultured in a medium containing minimum essential medium (MEM)-EAGLE Earle’s Salts Base (Biological Industries), penicillin (Hyclone), sodium pyruvate (PanReac AppliChem), and 10% fetal bovine serum (FBS; PEAK SERUM). The C₂BBe₁ cells were cultured in high-glucose DMEM (Biological Industries) with penicillin, sodium pyruvate, 10% FBS, and 0.01 mg/mL human transferrin (Sigma-Aldrich). The CCD841 CoN cells were cultured in ATCC-formulated Eagle’s MEM with 10% FBS. All the aforementioned cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂.

The human embryonic kidney epithelial HEK-293T cells and diploid lung IMR90 fibroblasts were purchased directly from the American Type Culture Collection (ATCC). The human cancer cell lines, including osteosarcoma U2OS cells, cervix carcinoma HeLa cells, prostate carcinoma DU-145 cells, and non-cancerous telomerase-immortalized foreskin BJ1 fibroblasts (BJ1-hTERT) were generously provided by Prof. Yosef Shiloh. Breast carcinoma MDA-MB-468 cells were a gift from Prof. Izhak Haviv. All cell lines, except for the IMR90 cells, were cultured in high glucose DMEM (Biological Industries, Beit-Haemek, Israel) supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine serum, and 1% (v/v) penicillin–streptomycin [34 (link),36 (link),37 (link),38 (link)]. IMR90 cells were grown in RPMI 1640 medium (Biological Industries) supplemented with 2 mM L-glutamine, 15% (v/v) fetal bovine serum, and 1% (v/v) penicillin–streptomycin.
The proteasomal inhibitor MG-132 and deubiquitinase (DUB) inhibitor N-ethylmaleimide (NEM) were purchased from Merck.
Smurf2-ablated (Smurf2KO) and wild-type control C57BL/B6 mice were housed at the SPF animal facility according to the FELASA guidelines and an experimental protocol approved by the Animal Care and Use Committee of Bar-Ilan University (BIU).

All cell lines were obtained from the Shanghai Institute for Biological Sciences (Shanghai, China). The human hepatoma cell lines (HCCL-M3, MHCC-97h, Huh7, SMMC-7721, HepG2, and HepG1), and the immortalized normal hepatocyte cell line (LO2) were cultured in high glucose DMEM (Biological Industries, Cronwell, CT, United States) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Cronwell, CT, United States). All media were supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (GibcoTM, Thermo Fisher Scientific, Waltham, MA, United States) and incubated at 37°C in a humidified chamber containing 5% CO₂.

HCC cell lines QGY‐7703, QGY‐7701, SMMC‐7721, and the normal hepatocyte cells HL‐7702 were purchased from Shanghai Institutes for Biological Sciences (China) and cultured in RPMI‐1640 medium (Biological Industries, USA) supplemented with 10% fetal bovine serum (Biological Industries, USA). HepG2 cells were maintained in high glucose DMEM (Biological Industries, USA). All the media were supplemented with 100μg/ml streptomycin and 100 U/ml penicillin (GibcoTM, Thermo Fisher Scientific, USA), and all cells were incubated at 37°C, 5% CO₂.

Human U2OS cells were maintained in low glucose DMEM (Biological Industries, Beit-Haemek, Israel), supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 1% Glutamine (Biological Industries, Beit Haemek, Israel), and 1% penicillin–streptomycin solution (Biological Industries). MCF7 and HeLa cells were maintained in high glucose DMEM (Biological Industries), supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution. All cells were grown at 37 °C in 5% CO₂. The GFP-dystrophin-MS2 U2OS cell line^{22 (link)} was selected using 165 μg/μl hygromycin (Sigma-Aldrich) and activated using Ponasterone A (PonA) at 5 μg/μl for 24 h (unless otherwise specified). Hyper-osmolarity treatment was performed with 1:100 10XPBS for 10 min. ATP depletion was performed using either 2-deoxy-d-glucose (20 mM, Sigma) or Na-azide (20 mM, Sigma) in medium for either 15 min or 1 h. Hoechst 33342 (2.5 μM) was used for labeling DNA. Puromycin (Invivogen) was used at a concentration of 10 μg/ml for 30 min in medium. Cycloheximide (Sigma-Aldrich) was used at a concentration of 100 μg/ml for 4 h in medium.

Previously established skin primary fibroblast cultures from the patient (informed consent was obtained, IRB #0485-09) [4 (link)], controls and human foreskin fibroblasts (HFF-1 ATCC) were maintained in high-glucose DMEM supplemented with 15% fetal bovine serum, L-glutamine, pyruvate and 50 µg/mL uridine (Biological Industries, Beit Ha’emek, Israel). For immunocytochemistry, cells were seeded on µ-slide 8 well ibiTreat sterile tissue culture slides (NBT; New Biotechnology Ltd., Jerusalem, Israel). For Western blotting or RNA analysis, cells were seeded, and grown in tissue culture bottles. For positive hypoxia controls in the immunostaining and Western blotting, cells were preincubated with 500 µL of CoCl₂ supplemented with a new fresh medium, for 6 or 24 h, at 37 °C and 5% CO₂ [10 (link)]. All the cells were incubated at 37 °C in a humidified 5% CO₂ atmosphere.