Ng methyl l arginine acetate l nmma

The RAW 264.7 macrophage cell line was obtained from the Institute of Biology of the Vietnam Academy of Science and Technology and cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 2 mM of L-glutamine, 10 mM of HEPES, and 1 mM of sodium pyruvate. It was supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in humidified air with 5% CO₂. RAW 264.7 macrophage cells in DMEM medium in 96-well plates were incubated for 24 h, and NO production was stimulated by LPS (1 μg/mL). Next, 100 μL of Griess reagent (50 μL of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 50 μL of 0.1% (w/v) N-1-naphthylethylenediamine dihydrochloride) was added, and it was incubated at room temperature for about 10 min. The Griess Reagent System from Promega Cooperation (USA) was used to determine the presence of nitrite. The microplate reader was used to assess absorption at 540 nm. N^G-Methyl-L-arginine acetate (L-NMMA) (Sigma) was used as a positive control at doses of 100, 20, 4, and 0.8 g/mL. The IC₅₀ values were calculated from non-linear regression analysis based on the dose–response curves using TableCurve 2Dv4 software. The experiments were repeated at least three times independently.

RAW 264.7 cells were seeded to a 96-well plate at a concentration of 2 × 10⁵ cells/well. After incubation for 24 h, the cultured medium was refreshed by DMEM without FBS. Then, the cells were treated with samples at different concentrations in the presence of LPS (1 µg/mL) for 24 h. N^G-Methyl-L-arginine acetate (L-NMMA) (Sigma) was used as the positive control and the cells treated with a diluted solution of DMSO (1.0%) were used as the negative control. Nitrite (NO₂^_), an indicator of NO production, was determined by the Griess reagent system (Promega Corporation, WI, USA). Briefly, 100 µL medium was mixed with 50 μL 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 50 μL 0.1% (w/v) N-1-naphthylethylenediamine dihydrochloride in a 96-well plate. The plate was then incubated at room temperature for 10 min. The optical density (OD) was measured at 540 nm by using a microplate reader (Biotek, Winooski, VT, USA). The concentration of nitrite in each well was calculated by using the NaNO₂ standard curve. The ability of NO inhibition of a sample was calculated by the following formula: (%) inhibition = 100%—(concentration of NO_sample/concentration of NO_{negative control}) × 100. The value of IC₅₀ (the half-maximal inhibitory concentration) was determined by using the Table Curve 2Dv4 software (Systat Software Inc., San Jose, CA, USA) [19 (link)].

Cearoin was isolated from the ethanol extract of dried heartwoods of D. odorifera as described previously [27 (link)]. Cearoin stock solution was prepared in DMSO and stored at −20 °C, which was diluted using serum-free media during the treatment. Similarly, stock solution of anticancer drug cyclosphosphamide was prepared in DMSO and diluted in serum-free media during treatment. Dulbecco’s Modified Eagle’s Medium (DMEM) and all medium additives were obtained from Gibco BRL. Cyclophosphamide, Sodium nitrite, N-Acetyl-l-cysteine (NAC), NG-methyl-l-arginine acetate (L-NMMA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and 2-7-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence kit were purchased from Sigma Aldrich (St Louis, MI, USA). PD98059 was purchased from Calbiochem (La Jolla, CA, USA). Caspase-3 activity kit was purchased from Biovison (Milpitas, CA, USA). The antibodies specific for α-spectrin, PARP, LC3B, p-ERK, ERK, p-JNK and JNK were purchased from cell signaling Technology (Danvers, MA, USA). The β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and GAPDH was purchased from Thermo Fisher (Illinois, Rockford, AL, USA).