2.3.2. Cytotoxicity

For cytotoxicity experiments, A549 cells were seeded in 96-well plates at a density of 1 × 10<sup>4</sup> cells per well and left to attach overnight. The cells were then treated with various concentrations (50–2000 μg mL−1) of aBC in the complete medium for 48 h. This was followed by the addition of 3-(4,5-dimethylthiazol-2-thiazyl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma Aldrich, St. Louis, MO, USA) solution to the final concentration of 1.0 mg mL−1. After 3 h of incubation, the medium was removed, the formed blue formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (Junsei Chemical Co., Tokyo, Japan) per well DMSO, and the absorbance at 570 nm (690 nm background control) was measured using a microplate reader (Biotek, Winooski, VT, USA). Low toxicity of aBC was observed in most treatments, with 75% of cells remaining viable after treatment with the highest dose (2.0 mg mL−1) of aBC for 48 h. Therefore, these treatment conditions were used for all subsequent experiments.

### 2.3.3. Endocytosis of aBC in A549 Cells

The aBC-exposed cells were harvested using 0.25% trypsin-EDTA (Gibco, Thermo Fisher Scientific, Walthem, MA, USA) from culture plates and cyto-centrifuged (800 raBC; 10 min) onto glass microscope slides using Cytospin 4 (Thermo Fisher Scientific, Walthem, MA, USA). Cell smears were fixed in methanol for 1 min and stained using Diff-Quik solution (Dade Diagnostics, Aguada, Puerto Rico). Stained cells were analyzed under a light microscope (Leica, Wetzlar, Germany).

### 2.3.4. Measurement of Reactive Oxygen Species (ROS) Levels

A549 cells were incubated for 30 min at 37 ◦C in RABCI 1640 medium containing 3.3 μmol L−<sup>1</sup> of 2,7-dichloro-fluorescein diacetate (DCF-DA) (Thermo Fisher Scientific, Walthem, MA, USA) for ROS detection. The cells were treated with aBC (2.0 mg mL−<sup>1</sup> for 48 h), then washed with Dulbecco's phosphate-buffered saline (DPBS; Welgene Inc., Daegu, Korea). The DCF-DA intensity in the cells was immediately measured at 495 nm (excitation)/529 nm (emission) using a microplate reader. ROS production in the cells is represented as a percentage of DCF-DA intensity relative to cell viability in each well, which was defined as 100%.
