Dcf da

ROS levels were determined using the ROS-sensitive dye 2, 7-dichlorofluoresceindiacetate (DCF-DA) (Nanjing Jiancheng Bioengineering Institute). HaCaT and WS1 cells were washed with PBS and incubated with DCF-DA (10 μM) for 30 min. The level of DCF fluorescence, reflecting the concentration of ROS, was measured by a fluorescence microscope. For skin tissues, the level of DCF fluorescence was measured at 488 nm using a 96-well plate reader.

Dulbecco's modified Eagle's medium (DMEM) was obtained from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was provided by Hangzhou Sijiqing Biological Engineering Materials Co. Ltd. (Hangzhou, China). Annexin V-FITC apoptosis detection kit was obtained from Kaiji Biotechnology (Hangzhou, China). The kits for determination of LDH, caspase 3, DHE, DCF-DA, 8-Oxo-dG, protein carbonyl, MDA, SOD, CAT, GSH-Px, and GSH were obtained from Jiancheng Bioengineering Institute (Nanjing, China). MitoSOX Red and Rh123 staining were obtained from Molecular Probes (Eugene, OR). Antibodies for cleaved caspase 3, Bcl-2, Bax, cytochrome C, UCP2, SOD2, NOX4, COX IV, P-FOXO3a, PCNA, BIM, P27KIP1, CAT, NRF1, TFAM, P-PGC-1α, PGC-1α, SIRT1, P-Akt, Akt, FOXO3a, acetylated lysine, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). All other reagents were purchased from Chinese suppliers.

Intracellular ROS levels were measured by using 2′,7′-dichlorofluorescein diacetate (DCFDA) (Nanjing Jiancheng Biology Engineering Institute, Nanjing, China) according to the manufacturer's protocol. The cells were incubated with 20 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA) for 45 min at 37°C and then washed two times with PBS. The cells were collected and analyzed by using a microplate reader (excitation/emission: 500/525 nm).