Cell Cultures, Transfection, and Addition of Sirt1 Activator

The HLE cell line SRA 01/04 was used in this study (the STR profile for this cell line is shown in Supplemental Material 4 ). The HLE cells used for the evaluation of ROS formation, apoptosis ratio, and oxidative and antioxidant contents were seeded in six-well cell culture plates, and the HLE cells used for western blotting were seeded in 100*20-mm cell culture dishes. The cells were cultured in DMEM-LG containing 10% FBS and 1% penicillin/streptomycin. The cells were incubated at 37°C and 5% CO₂ and grown to 70% confluence for the experiments.

To assess the function of increased LSS expression in HLE cells exposed to UV-B, LSS was overexpressed in the experimental group. Lentiviruses containing the LSS overexpression sequence were purchased from GeneChen and transduced into HLE cells according to the manufacturer’s instructions, and these transduced cells were used as the experimental group. Some HLE cells were transfected with lentivirus containing a meaningless sequence and used as the control group (MOI = 50). The fluorescence-positive cells were observed by microscopy (fluorescence-positive cells obtained with different MOIs are shown in the Supplemental Figure 1A in Supplemental Material 1 ), and a western blot analysis of LSS was performed to assess the transfection efficiency (the protein bands obtained in the western blots are shown in the Supplemental Figure 1B in Supplemental Material 1 ).

To further explore whether the increase in LSS was related to a decrease in Sirt1 in HLE cells exposed to UV-B, we included cells pre-treated with resveratrol as an experimental group. A stock solution of resveratrol was obtained by dissolving resveratrol into absolute ethanol to a concentration of 100 mM. HLE cells were seeded in culture medium containing 50 μM resveratrol for 24 h before UV-B exposure. Twenty-four hours after the cells were exposed to UV-B, the expressions of Sirt1, SREBF2, HMGCR, and LSS proteins were evaluated by western blott.