3.3.2. Cell Viability Assay

The cytotoxicity of LJSF4 on HaCaT cells were determined by MTT assay. In brief, HaCaT cells were seeded in a 96-well plate at a density of 3 × 10<sup>4</sup> cells per well. After 24 h, the supernatant was discarded and wells were treated with 25, 50, 100 μg/mL of LJSF4, respectively. Then, after being further incubated for 24 h, the MTT solution was applied into the wells for 4 h. At last, the formazan crystals were dissolved in DMSO, and the absorbance were measured at 540 nm by an ELISA plate reader (BioTek PowerWave XS, Winooski, VT, USA) [10].

#### 3.3.3. Measurement of Intracellular ROS Generation and Cell Viability in UVB-Irradiated HaCaT Cells

Intracellular ROS generation of LJSF4 on UVB-irradiated HaCaT cells was determined by the DCFH-DA method [44]. Briefly, after incubation for 24 h, cells were treated with LJSF4 for 30 min. Then, 500 μg/mL of DCFH-DA was added into each well and incubated for 30 min. Subsequently, cells were exposed to 30 mJ/cm<sup>2</sup> of UVB and measured according to the reported procedure of Wang et al. [42].

In order to measure the cell viability of LJSF4 on UVB-induced HaCaT cells, cells were treated with LJSF4 (25, 50, 100 μg/mL). After being cultured for 2 h, cells were exposed to 30 mJ/cm<sup>2</sup> of UVB and incubated for 24 h. Subsequently, cell viability was examined by the MTT assay described previously [10,45].

#### 3.3.4. Measurement of Apoptosis in UVB-Irradiated HaCaT Cells

As in the procedure described by Naito et al., the apoptosis body formation was assessed by nuclear staining [46]. After being seeded in 24-well plates for 24 h, cells were treated with LJSF4 and incubated for another 2 h. Then, cells were exposed to 30 mJ/cm<sup>2</sup> of UVB and incubated with serum-free DMEM solution for 6 h. After incubation, cells were treated with Hoechst 33342 (stock, 10 mg/mL) for 10 min. At last, the stained cells were photographed by a fluorescence microscope equipped with a Cool SNAP-Procolor digital camera (Olympus, Tokyo, Japan). Apoptosis levels were measured using Image J software automatically.
