Dulbecco s modified eagle s medium high glucose

Basal medium consisted of Dulbecco’s modified Eagle’s medium-high glucose (Sigma-Aldrich, USA) supplemented with 5 mM sodium bicarbonate (Cinética Química Ltda, Brazil), penicillin (100 units/mL; Sigma-Aldrich), streptomycin (0.1 mg/mL; Sigma-Aldrich), amphotericyn B (0.25 μg/mL; Sigma-Aldrich), gentamicin (60 mg/L; Schering-Plough, USA), and 10% of either aHS or FBS (Cripion Biotecnologia Ltda, Brazil). Adipogenic medium consisted of basal medium with 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), 200 μM indomethacin (Sigma-Aldrich), 1 μM dexamethasone (Aché, Brazil), and 10 μM insulin (Eli Lilly and Company, USA). Osteogenic medium consisted of basal medium with 50 μg/mL ascorbate-2-phosphate (Ecibra, Brazil), 10 mM β-glycerophosphate (Sigma-Aldrich), and 0.1 μM dexamethasone (Aché). Chondrogenic medium consisted of basal medium with 1 mM dexamethasone (Aché), 125 μg/mL bovine serum albumin (PAA, Austria), 1 mM pyruvate (Sigma-Aldrich), 200 U/mL insulin (Eli Lilly and Company), 3.25 μg/mL transferrin (Wako, Brazil), 0.01 μg/mL transforming growth factor-β1 (Sigma-Aldrich), 5 mg/mL ascorbate-2-phosphate (Ecibra) and a reduced concentration of serum supplements - 1% aHS or 1% FBS [5 (link)].

C9-ALS patient fibroblasts were kindly provided by Dr Aaron Gitler (Stanford University). HEK293T cells (ATCC) and C9-ALS patient fibroblast were maintained under 37 °C with 5% CO₂ in Dulbecco’s modified Eagle’s medium–high glucose (Sigma Aldrich) supplied with 10% fetal bovine serum. Transfections were performed according to manufacturer’s instructions. Plasmids transfections were carried out by using Lipofectamine 3000 (cat#: L3000015, Invitrogen), and siRNA knockdown experiments were performed using Lipofectamine RNAi-MAX reagent (cat#: 13778150, Invitrogen). pCDNA3-Flag-GR80 was described before (39 (link)). After 72 h transfection, cells were washed with 1× PBS and then subjected to lysis and Western blot analysis. siRNAs used in the study were purchased from Invitrogen: siCon (cat#: 12935-400), siAKT1 (VHSS40082), siAKT2 (VHS41339), and siAKT3(cat#: AM51331).

This study analyzed 32 lateral menisci from 29 patients (mean age, 75.1 years; range, 59–90 years; 5 men and 24 women) with knee OA or osteonecrosis who underwent total knee arthroplasty. The meniscus was processed within 48 h after surgery and divided into six samples (A, Am, Ma, Mp, Pm, and P) for histological evaluation (Fig. S5A), quantitative assessment, gene expression analysis, and protein secretion analysis. For the compression test, the samples were drilled using a disposable biopsy punch (diameter = 5 mm; Kai Medical, Solingen, Deutschland). For protein secretion analysis, each sample was cut in half vertically, where one half was cultured in Dulbecco’s modified Eagle’s medium/high glucose (Sigma-Aldrich, St. Louis, MO, USA) with 1% penicillin/streptomycin, and incubated at 37 °C, 90% humidity, and 5% CO₂. The other half was used for histological and gene expression analyses. Except for protein secretion analysis, all samples were frozen at − 80 °C until use.

Human adipose tissue was harvested from healthy patients who had abdominal reduction surgery. All the donors provided written informed consent. The approval number in the Ethics Committee in Research from Federal University of Minas Gerais is—ETIC 0023.0.203.000-11. The hASCs were obtained and cultured as described previously by Zuk et al. [18 (link)]. The cells were cultured in basal medium Dulbecco's modified Eagle's medium-high glucose (Sigma-Aldrich) supplemented with 5 mM sodium bicarbonate, penicillin (100 units/mL), streptomycin (0.1 mg/mL), amphotericin B (0.25 μg/mL) (Sigma-Aldrich), gentamicin (60 mg/L, Schering-Plough), and 10% of fetal bovine serum (FBS) (Cripion Biotecnologia LTDA, Brazil) at 37°C in a 5% CO₂ humidified atmosphere. And the hASCs were cultured under basal medium with SWCNT-MA suspension (diluted 1/5 or diluted 1/10 in basal medium) to perform the assays.

HaCaT cells (immortalized human keratinocytes) were obtained from CLS Cell Lines Service (Eppelheim, Germany) (Boukamp et al., 1988 (link)) and cultured as described previously (Saika et al., 2021 (link)). Cultured HaCaT cells were seeded at a concentration of 5 × 10⁵ cells/10 mL in Dulbecco’s modified Eagle’s medium high glucose (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin with 100 μg/mL streptomycin (Nacalai Tesque) and incubated for 24 h at 37°C in 5% CO₂. The medium was then replaced with Dulbecco’s modified Eagle’s medium without FBS, and the cells were treated with 3 µM mead acid or vehicle (ethanol) for 30 min before stimulation with HB-EGF (1 ng/mL, PeproTech, Cranbury, New Jersey, United States) for 1 h at 37°C in 5% CO₂.

Penicillin-streptomycin solution (PS), trypsin-EDTA solution, dimethyl sulfoxide (DMSO), thiazolyl blue tetrazolium bromide (MTT), and Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from SAFC Biosciences (Victoria, Australia). Methanol (ACS Grade), ethyl acetate (ACS Grade), dichloromethane (ACS Grade), and n-hexane (ACS Grade) were purchased from Mallinckrodt (St. Louis, MO, USA). n-Butanol (ACS Grade) was purchased from J. T. Baker. Methanol-d₄ (CD₃OD), acetone-d₆ (CD₃COCD₃), and chloroform-d (CDCl₃) were purchased from Merck (Darmstadt, Germany).