Cell cycle detection kit

Cell cycle analysis was performed using the Cell Cycle Detection Kit (Keygentec). A549 and BESA2B cells (1 × 10⁶) were harvested, washed with PBS twice and fixed in 500 μL 70% ice‐cold ethanol for 2 hours at 25°C. The cells were then washed twice with cold PBS and incubated in PI (400 μL) and RNase (100 μL) for 30 minutes at 37°C in the dark. The PI signal was detected via FCM (BD Biosciences). The percentages of cells in the G1, S and G2 phase were determined and compared. Each experiment was repeated three times.

Crystal violet (Macklin, 548‐62‐9, Shanghai, China) was used for Crystal violet staining. 5‐Ethynyl‐20‐deoxyuridine (EDU) (100 mM) (BeyoClickTM EdU‐555, C0075S, China) was used for detecting the percent of cell proliferation. Annexin V‐FITC/PI apoptosis detection kit (Vazyme, A211, China) was performed, according to the manufacturer's instruction. Cell cycle detection kit (Keygentec, KGA511, China) was used for detecting cell cycle. The JC‐1 apoptosis assay kit (Keygentec, KGA601, China) was used according to the kit protocol. Reactive oxygen species assay kit (Beyotime, S0033, China) was used to detect the ROS level of cells. The r‐protein A/G magenetc IP/Co‐IP kit (ACE, China) was used to Process the COIP samples. For more detailed steps refer to the attached Supplementary Methods and Materials.

To determine cell cycle distribution, the cells were plated in 6-well plates and transfected with miRNA mimics or siRNA duplexes. After transfection, the cells were harvested, treated with cell cycle detection kit (keygentec Nanjing China) and tested using a FACSCalibur instrument (Becton Dickinson, CA, USA). The data were analyzed using the CellQuest Pro software (BD Biosciences).