Fluorescence spectrophotometer

Intracellular ROS levels were measured using a Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, China) according to the manufacturer’s instructions. Briefly, stable cells expressing sh-TOMM20 and sh-Control were incubated with DCFH-DA at 37 °C for 20 min, and then were observed with a fluorescence microscopy (Olympus). The fluorescence was measured at 488 nm excitation and 525 nm emission by a fluorescence spectrophotometer (BD Bioscience).

CCK8 method was used to analyze the effects of different concentrations of U73122, MK-2206 (2HCI), and PTX on keratinocytes. Briefly, the cells were seeded in 96-well plates at a density of 1.2 × 10⁴ cells per well. After 24 h incubation, cells were respectively treated with various concentrations of PTX (100 ng/ml, 150 ng/ml, 200 ng/ml, 250 ng/ml, 300 ng/ml, and 350 ng/ml) for 4 h or U73122 (4.5 μM, 9 μM, 13.5 μM, 18 μM, 22.5 μM, and 27 μM) for 30 min or MK-2206 (2HCI) (3 μM,6 μM, 9 μM,12 μM, 15 μM, 18 μM, 21 μM, and 24 μM) for 2 h. Following the treatment, 10 μl CCK8 was added to each well, and 2 h later, absorbance was tested at a wavelength of 405 nm using a fluorescence spectrophotometer (BD Biosciences, San Jose, CA).