*2.9. Western Blot Analysis*

B16F10 cells were treated with FTGML (0–1.6 mg/mL) or kojic acid (0.75 mg/mL) for 48 h. Lysis buffer (150–250 μL) was added to each well to complete the lysis. Then, the lysed samples were centrifuged at 12,000× *g* for 15 minutes at 4 ◦C, and the supernatant was obtained for protein quantification (BCA assay kit, PICPI23223, Thermo Fisher Scientific, Carlsbad, CA, USA), and stored in a refrigerator at −80 ◦C for the next analysis.

An equal amount of protein (20 μg/sample) was bathed in boiling water for 10 min after being mixed with 5 × SDS loading buffer, separated by 12% SDS polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, Carlsbad, CA, USA). The PVDF membrane was blocked in 5% skim milk in TBST buffer (PBS containing 0.05% Tween-20) for 1 h at room temperature. The membranes were incubated with several antibodies overnight at 4 ◦C after a short wash. These antibodies include anti-MITF (1:500), anti-GAPDH (1:2500), anti-p38 (1:1000), anti-p -p38 (1:1000), anti-p-JNK (1:200), anti-JNK (1:500), anti-p-PI3K (1:500), anti-PI3K (1:500), anti-p-Akt (1:2000), and anti-Akt (1:1000). After incubation, the membrane was washed thoroughly with TBST buffer, and further incubated with goat anti-rabbit HRP-labeled secondary antibody (1:1000, Beyotime Biotechnology Co., Ltd., Shanghai, China) at 37 ◦C for 1 h. The blots were visualized using enhanced chemiluminescence (ECL), and quantified by Image-pro plus software (Media Cybernetics, Rockville, MD, USA).

### *2.10. Statistical Analysis*

All results were expressed as mean ± standard deviation. SPSS version 20 (SPSS Inc., Chicago, IL, USA) was used for analysis of variance (*p* < 0.05). The Duncan multirange test was used for the comparison of means. Three replicates were used for each analysis. Principal component analysis (PCA) was used to analyze the correlation between the different properties of anti-melanin and anti-oxidation. The above indicators were used as active variables, and the concentration of FTGML was used as the observed value. Two-dimensional or three-dimensional graphs were drawn using Origin Pro 2018 software. Hierarchical cluster analysis (HCA) was used to visualize and emphasize the similarities between individuals. The difference is usually expressed by the distance between individuals [19]. Origin Pro 2018 software was used to determine the correlation between indicators by the Pearson correlation coefficient in binary linear correlation.

#### **3. Results and Discussion**

#### *3.1. Effects of FTGML on B16F10 Cells Viability*

B16F10 cells were treated with 0.1–1.6 mg/mL FTGML and 0.75 mg/mL kojic acid for 0 h, 24 h, and 48 h, respectively. The cell viability is expressed as a percentage relative to the cells in the blank control group (cells without any drugs), and the results are shown in Figure 1. FTGML and kojic acid showed low toxicity to B16F10 cells at all treatment concentrations (cell viability > 80%, [20]). Based on these results, the concentration of FTGML in the range of 0.1 mg/mL to 1.6 mg/mL can be used for subsequent experiments.

**Figure 1.** Effects of FTGML and kojic acid (KA) on cell viability. The CCK8 method was used to detect the effects of FTGML at 0, 0.1, 0.2, 0.4, 0.8, and 1.6 mg/mL; and kojic acid at 0.75 mg/mL on the activity of B16F10 cells (Note: \*, *p* < 0.05).

#### *3.2. Effects of FTGML on Apoptosis Rate of B16F10 Cells*

Apoptosis is a basic biological phenomenon of cells, and plays an essential role in the removal of unwanted or abnormal cells by multicellular organisms. The disorder of apoptosis may be directly or indirectly related to the occurrence of many diseases, such as tumors and autoimmune diseases. Therefore, an apoptosis assay is often used to evaluate the development and application potential of active ingredients in food in the field of functional food [21].

The effect of 0.1~1.6 mg/mL FTGML treatment for 48 h on the apoptosis of B16F10 cells is shown in Figure 2. It can be seen from Figure 2A that as the concentration of FTGML increased, the proportion of normal living cells gradually decreased, and the proportion of early apoptotic cells, late apoptotic cells, and necrotic cells increased. Figure 2B shows the apoptosis rate of B16F10 cells (i.e., the total proportion of early apoptotic cells to late apoptotic cells). It can be seen from the figure that FTGML treatment promoted the apoptosis of cells, and the apoptosis rate was positively correlated with the concentration of

FTGML. There was no significant difference in apoptosis rate between B16F10 cells treated with kojic acid (0.75 mg/mL) and FTGML at high concentration (≥0.8 mg/mL) (*p* > 0.05).

**Figure 2.** Effects of FTGML and kojic acid (KA) on cell apoptosis. (**A**) The proportion of different states (normal cells, early apoptotic cells, late apoptotic cells, and necrotic cells). (**B**) The apoptosis rates (the sum of the proportions of early and late apoptosis) of B16F10 cells at different treatments. (**C**) The effect of FTGML and kojic acid on the expression of STAT3. Means with different lowercase letters are significantly different (*p* < 0.05) among the different groups.

STAT3 (signal transducer and activator of transcription 3) is both a cytoplasmic signal molecule and a nuclear transcription factor, which is involved in cell proliferation, transformation, and migration. At present, STAT3 has been identified as a major oncogene in the development of melanoma [22]. There is also genetic evidence for a direct role of STAT3 in melanoma cell transformation [23]. As can be seen from Figure 2C, when the concentration of FTGML was greater than 0.4 mg/mL, the ratio of phosphorylated STAT3 (p-STAT3) to STAT3 was lower than 1, and significantly lower than that of the blank group (*p* < 0.05). This suggests that medium-high concentrations (≥0.4 mg/mL) of FTGML can reduce the activation/phosphorylation of STAT3. Some researchers have found that increasing the activation/phosphorylation level of STAT3 can promote the growth of melanoma, whereas silencing STAT3 can significantly inhibit the proliferation of melanoma cells, and promote cell apoptosis [22]. This is consistent with the results shown in Figure 2B.
