4.3. Cell Morphological Examination and Viability Assay

The A375.S2 cells (1 × 10⁵ cells/well) were seeded onto 12-well plates overnight with a MEM culture medium and they were incubated with berberine at final concentrations (0, 1, 1.5, and 2 μM) in triplicate for 24 h. After treatment, the cells were examined and photographed under contrast-phase microscopy at 200×. The cells were harvested, washed with PBS, and were stained with PI (5 μg/mL) for measuring the total percentage of cell viability by using flow cytometry (Becton-Dickinson, San Jose, CA, USA) as described previously [62,63].

The A375.S2 cells (1 × 10⁵ cells/well) were treated with various concentrations (5, 10, or 15 μM) of PLX4032, an inhibitor of the BRAF^V600E mutation, for 48 h and the cells were harvested to measure the total viable cell number as described previously [62,63]. To calculate the IC₅₀ of the PLX4032, 6 μM of A375.S2 cells were used to generate the PLX4032-resistant A375.S2 cells, as described previously with modifications [64]. The resistant A375.S2 cells were incubated with various concentrations (0, 5, 10, or 15 μM) of PLX4032 for 48 h to measure the total viable cell number. The resistant A375.S2 cells were treated with various concentrations (2, 4, and 6 μM) of berberine for 48 h and were harvested for measuring the total viable cell number as described previously [62,63].