Cell culture reagents

Myxothiazol (Myxo), mito-TEMPO (mitoTP), standard chemicals, and cell culture reagents were purchased from Euroclone s.r.l. (Milan, Italy), unless otherwise indicated. Terpestacin was kindly provided by Dr. HoJeong Kwon (Yonsei University, South Korea).

[³H]-DTG (29 Ci/mmol) was purchased from PerkinElmer Life Sciences (Zavantem, Belgium), DTG was purchased from Tocris Cookson Ltd. (U.K.) and (+)-Pentazocine was obtained from Sigma-Aldrich-RBI s.r.l. (Milan, Italy). Wistar Hannover rats (250–300 g) were retrieved from Harlan, Italy. Cell culture reagents were purchased from EuroClone (Milan, Italy). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) was obtained from Sigma-Aldrich (Milan, Italy). [10-(2,5-dihydroxy-3,4-dimethoxy-6,ethylphenyl)decyl]triphenyl-phosphonium, monomethanesulfonate (mitoquinol or mitoQ) was obtained from Cayman Chemical (Ann Arbor, MI, USA).

Cell culture reagents were purchased from Euroclone (Milan, Italy). COX (ovine/human) inhibitor screening assay kit (Catalog No. 560131, Cayman Chemicals, Ann Arbor, MI, USA) was purchased from Sigma-Aldrich, Burlington, MA, USA. Mofezolac was prepared in our laboratory [29 ]. The other reagents were purchased from Sigma-Aldrich, Burlington, MA, USA.

The materials and reagents used in the synthetic procedures were purchased from Sigma Aldrich Co. (Stenheim, Germany) and used without purification. All solvents were analytically pure and dried before use. TLC was carried out on aluminum sheets precoated with silica gel 60 F254 (Merck). Column chromatography was performed using silica gel 60 (230–400 mesh).
High-resolution MS (HRMS) ESI analyses were performed on a Xevo G2-XSQTof (Waters) mass spectrometer. Mass spectrometric detection was performed in the in the positive ion mode. The ¹H and ¹³C NMR spectra were recorded at 400 and 100 MHz, respectively, on an Agilent Technologies 400 MHz Premium Shielded spectrometer. Chemical shifts (δ) are reported in ppm relative to TMS and coupling constants (J) in Hz. Cell culture reagents were obtained from Euroclone (Milan, Italy). Chemical reagents and propidium iodide (PI) were obtained from Sigma Aldrich (St. Louis, MO, USA). The Annexin V-FITC apoptosis detection kit was obtained from Biolegend (San Diego, CA, USA). Chemiluminescent substrate and secondary antibody (32460) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Bradford reagent and polyvinylidene difluoride (PVDF) membranes were obtained from Bio-Rad (Hercules, CA, USA).

Cell culture reagents were purchased from EuroClone (Milan, Italy). Protoporphirin IX (Hemin), trizma base, potassium chloride, imidazole, sodium citrate tribasic dihydrate and lithium chloride, Amicon Ultra-4 10 kDa MWCO centrifugal filter device was purchased from Merck. Nickel-NTA agarose beads (low density), n-octyl β-D-glucopyranoside (β-OG) and phenylmethylsulfonyl fluoride (PMSF) were from Gold Biotechnology. Hexaethylene Glycol Monodecyl Ether (C₁₀E₆) detergent was purchased from Anatrace (Maumee, OH). Phusion High Fidelity (HF) DNA was from Biolabs. Sf-900 II SFM medium, HALT protease inhibitor single-use cocktail EDTA-free, Pierce BCA protein assay kit, Snakeskin dialysis tubing (10 kDa MWCO, 35 mm), and glycerol were purchased from Thermo Fisher Scientific Italia (Monza, Italy). TGX 10% precast polyacrylamide gels and all reagents for SDS-PAGE were purchased from BIO-RAD Laboratories Srl (Milan, Italy). Tobacco Etch Virus (TEV) protease was produced as described^{33 (link)}. Oxygraph buffer: 200 mM Tris–HCl (pH 8.3), 2 M KCl, 1 mM phenol.

MSCs were harvested from the bone marrow of femurs and tibias of the Mecp2^+/− and WT mice euthanized as above reported. We inserted a 21-gauge needle into the shaft of the bone and flushed it with Minimal Essential Media with alpha modifications (α-MEM). Then, we collected and plated cells from each mouse onto two 100-mm dishes with α-MEM containing 10% fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF). After 24 to 48 h, non-adherent cells were discarded while adherent ones were washed twice with phosphate-buffered saline (PBS). Cells were incubated for 7 to 10 days in proliferating medium to the confluence and then propagated to conduct experiments at passages 3 or 4. Cells were grown in monolayers and maintained at 37 °C under 5% CO₂. The cell culture reagents were purchased from EuroClone S.p.A, Pero, Italy.