*4.3. Cell Viability*

The cytotoxic effects of juglone were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, J774.1 cells were seeded in 24-well plates (2 × 10<sup>5</sup> cells/well) in 500 μL of cell culture medium and incubated in a 37 ◦C and 5% CO2 incubator for 12 h to allow for cell adhesion. After incubation, cells were treated with increasing concentrations of juglone (3.1–50 μM) for 24 h. Subsequently, the cell culture medium was replaced with phenol red-free medium, and 50 μL of 5 mg/mL MTT solution was added to each well. Cells were incubated with MTT for an additional 4 h in a 37 ◦C and 5% CO2 incubator. The supernatant was removed, and 200 μL dimethyl sulfoxide (DMSO) was added to each well to dissolve the MTT crystals. Optical absorbance was measured at 450 nm using a microplate reader (Marshall Scientific, Hants, UK). Cell viability was calculated, according to the following Formula (1):

Cell viability (%) = (Abs 450 nm of untreated cells − Abs 450 nm of treated cells/Abs 450 nm of untreated cells) × 100 (1)

#### *4.4. Measurement of Nitric Oxide (NO)*

To evaluate the anti-inflammatory effects of juglone in macrophages, NO production was measured in J774.1 cells. The amount of NO produced in response to LPS stimulation was measured using the Griess test, which determines the amount of nitrite (NO2−) generated. J774.1 cells were seeded in 24-well plates (2 × 10<sup>5</sup> cells/well) and incubated at 37 ◦C for 12 h to allow for cell adhesion. After incubation, the cell culture medium was replaced with phenol red-free medium. Cells were treated with different concentrations of juglone for 2 h, followed by stimulation with LPS (1 μg/mL) for 24 h at 37 ◦C and 5% CO2. After incubation, 200 μL of the cell culture solution and 50 μL of Griess reagen<sup>t</sup> [1% (*w*/*v*) sulfanilamide and 0.1% N-1-naphthylethylene diamine in 2.5% (*v*/*v*) phosphoric acid] were mixed in a 96-well plate. After a 10 min incubation at room temperature, the absorbance was measured at 540 nm. The standard calibration curve was obtained by measuring the optical absorbance

of serial dilutions of sodium nitrate (NaNO2). The amount of NO produced was determined using the standard calibration curve.

#### *4.5. Measurement of the Antioxidative Capacity of Juglone*

The antioxidative capacity of juglone was measured using the hydrophilic peroxyl radical scavenging capacity (PSC) assay [36]. To measure ROS in J774.1 cells, 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma, St. Louis, MO, USA) was used. The cells were seeded into 24-well plates (2 × 10<sup>5</sup> cells/well) and incubated for 12 h. Cells were treated with 2.5, 5, and 10 μM juglone. After 2 h of incubation, cells were exposed to 1 μg/mL LPS for 24 h in a 37 ◦C and 5% CO2 incubator. After incubation, the cells were washed twice with cold phosphate-buffered saline (PBS) and incubated with 10 μM of DCFH-DA for 30 min at 37 ◦C. The fluorescence intensity was measured on a fluorescence microplate reader (Marshall Scientific) using an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
