β mercaptoethanol

For DNA extraction, we used an optimized cetyl trimethyl ammonium bromide (CTAB) method (TCM-CTAB)^{47 (link)}. Each sample (1.0 g) was completely dissolved with 0.1 M Tris–HCl, 20 mM EDTA (pH 8.0, 2 mL). Dissolved solution (0.4 mL) was diluted with extraction buffer (0.8 mL) consisting of 2% CTAB; 0.1 M Tris-HC1 (pH 8.0); 20 mM EDTA (pH 8.0); 1.4 M NaCl, and then 100 μL 10% SDS, 10 μL 10 mg/mL Proteinase K (Sigma, MO, USA) and 100 μL β-Mercaptoethanol (Amresco, OH, USA) were added and incubated at 65 °C for 1 h with occasional swirling. Protein was removed by extracting twice with an equal volume of phenol:chloroform:isoamyl-alcohol (25:24:1), and once with chloroform: isoamyl-alcohol (24:1). The supernatant was incubated at − 20 °C with 0.6 folds of cold isopropanol for 30 min to precipitate DNA. The precipitate was washed with 75% ethanol, dissolved and diluted to 10 ng/μL with TE buffer, and then used as a PCR amplification template. DNA concentration was quantified on Qubit2.0 Fluorometer.

H9 hESCs (WiCell Institute, Madison, WI, USA, passages 25–55) were cultured on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs) as described in a standard protocol (http://www.wicell.org). The hESC culture medium, consisting of Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco), 20% Knockout serum replacement (Gibco), 0.1 mM β-mercaptoethanol (Amresco), 1% NEAA (GIBCO), 0.5% l-glutaMAX (Gibco) and 4 ng/ml FGF-2 (Sino Biological).

R1/E mESCs was obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and maintained as described in our previous studies29 (link)63 (link). In brief, mESCs were grown at an optimal density in gelatin (Sigma) coated dishes without feeder cells in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) supplemented with 10% fetal bovine serum (FBS; HyClone), 2 mM glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 1× nonessential amino acids (NEAA; Invitrogen), 55 μM β-mercaptoethanol (Amresco), and 1 × 103 units/mL recombinant leukemia inhibitory factor (LIF; Sigma) in an environment containing 5% CO₂ at 37 °C. The medium was changed every second day.

The activities of NR and GS were determined according to Zhao et al. [102 ]. The activity of GOGAT was determined by the method of Singh and Srivastava [103 (link)] with some modification. The extraction buffer contained 1 mM EDTA (Amresco, Solon, OH, USA), 1 mM β-mercaptoethanol (Amresco, Solon, OH, USA), 1 mM MgCl₂ (Richjoint Chemical, Shanghai, China), 10 mM Tris (Amresco, Solon, OH, USA)-HCl (pH 8.2). The assay buffer contained 0.4 mL 20 mM l-glutamine (Sigma-Aldrich Co., St. Louis, MO, USA), 0.5 mL 20 mM α-oxoglutarate (Sigma-Aldrich Co., St. Louis, MO, USA), 0.1 mL 10 mM KCI (Richjoint Chemical, Shanghai, China), 0.2 mL 3 mM NADH (F. Hoffmann-La Roche Ltd., Basel, Switzerland) and 0.3 mL of the enzyme preparation. The final volume was completed to 3.0 mL with 25 mM Tris-HCl buffer (pH 7.6) [103 (link)]. The reaction was started by the addition of l-glutamine immediately following the enzyme preparation. The absorbance decrease was recorded for 4 min at 340 nm by using a UV-1800 spectrophotometer (Shimadz, Kyoto, Japan). The activity of GOGAT was expressed as the amount of NADH oxidized per minute per milligram protein.

Total RNAs were separately isolated from the decapsulated testes of four transgenic adult mice. Following homogenization in 0.5 ml RLT buffer (Qiagen) supplemented with 1.5% β-mercaptoethanol (Amresco) with a PRO Scientific 200 homogenizer (PROScientific Inc., Oxford, CT), RNAs were extracted as described62 (link)63 (link). Total RNAs were DNase treated (Turbo DNase, Ambion) and resolved using the 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Prepared mouse total testes RNAs64 (link) were used to construct individual RNA-seq libraries according to established protocols63 (link). Briefly, pre-amplified cDNA libraries were generated from 5 ng of total testis RNA using the Seq-plex system (Sigma) and used to construct sequencing libraries (DNA Ultra-Low, NEB). RNA-seq libraries were subjected to paired-end sequencing on the Illumina Hi-Seq 2500 platform, as above.

BmEos were generated as described elsewhere [21 (link)]. Briefly, bone marrow cells were flushed from the femora and tibiae of 4-week-old BALB/c mice and cultured in RPMI-1640 medium (Welgene, Gyeongsan, South Korea) containing 20% fetal bovine serum, 100 IU/mL penicillin, 10 ug/mL streptomycin, 2 mM glutamine, 25 mM HEPES, 1 × nonessential amino acids, 1 mM sodium pyruvate (Life Technologies, Grand Island, NY, USA), 50 μM β-mercaptoethanol (Amresco, Solon, OH, USA) and supplemented with 100 ng/mL stem-cell factor (PeproTech) and 100 ng/mL FLT3-Ligand (PeproTech) from days 0 to 4. From day 4, cell medium was replaced with medium containing only 10 ng/mL IL-5 (PeproTech). On day 14, bmEos were incubated with PE-conjugated rat anti-mouse Siglec-F (BD Biosciences) and APC-conjugated anti-mouse CCR3 (R&D Systems, Minneapolis, MN, USA). Data were acquired using the FACSCalibur flow cytometer (BD Biosciences) and analyzed using Cell Quest Prosoftware (ver. 5.2.1 BD Biosciences). Siglec-F and CCR3-positive cells were identified by comparison with PE-conjugated IgG2a, κ isotype, and APC-conjugated IgG2a isotype controls.

Apatinib (purity ≥ 98%, Jiangsu Hengrui Pharmaceutical Co., Ltd.), vemurafenib (purity ≥ 98%, Selleckchem, USA), vemurafenib raw materials (purity ≥ 98%, Biedue, Shanghai). RPMI-1640 culture medium (Hyclone, USA), fetal bovine serum (Invitrogen, USA), double antibody (Invitrogen, USA), 0.25% trypsin (Invitrogen, USA). CCK8 Assay Kit (Tongren Chemical, Japan), Annexin V-FITC/PI Apoptosis Detection Kit (BD, USA), DMSO (Sigma, USA). BCA Protein Assay Kit (Pierce, USA), M-PER Cell Extraction Reagent (Pierce, USA). MEK/p-MEK antibody, ERK/p-ERK antibody (Abeam, USA), hypersensitive ECL chemiluminescence detection kit (Thermo, USA), polyvinylidene difluoride membrane (PVDF membrane) (Millipore, USA), Matrigel (BD, USA). sodium carboxymethyl cellulose (CMC-Na) (Sinopharm Group, Beijing), hydroxypropyl cellulose (Klucel LF) (Changwei Medicine, Shanghai), DMSO (GmbH, Germany), Tris-Tris (Hydroxymethyl) aminomethane, hydrazone red S, Tween-20, and β-mercaptoethanol were all chemical analysis reagents purchased from Amresco, USA.