Edu kits

Cells were inoculated in a 96‐well plate at a density of 1.6 × 10⁵ cells/well and cultured for 48 hours. After culture, EdU assay was performed in accordance with the protocols of EdU kits (C10310; Guangzhou RiboBio Co., Ltd.). Cells in each well were cultured with 100 μL of 50 μmol/L EdU at 37°C for 4 hours, fixed with 4% formaldehyde at room temperature for 15 minutes and treated with 0.5% Triton X‐100 at room temperature for 20 minutes for permeabilization. After that, cells in each well were incubated with 100 μL of Apollo^® compound (C10338‐2; RiboBio Company) at room temperature for 30 minutes, stained using 100 μL of hoechst33342 (Ribobio Company) for 30 minutes and observed under a fluorescence microscope (Olympus). The number of EdU positive cells (red blood cells) was calculated using the Image‐Pro Plus 6.0 software (Media Cybernetics).17

HL‐60, K562 and THP‐1 cells (2.0 × 10⁵ cells/mL) were exposed to 0.3125–5 µM ASK for 24 h. The cells treated without ASK were employed as the control group. Cell proliferation was measured using EdU kits (RIBOBIO, Guangzhou, China) according to the manufacturer's instructions to analyse the incorporation of EdU during DNA synthesis. Cells were collected by centrifugation and incubated with 50 μM EdU for 2 h. The supernatant was discarded after the cells were collected by centrifugation. Wash cells with PBS for one time, and then fix cells with methanol for 30 min. The cell membranes were lysed with 0.5% Triton X‐100 in a shaking table. Then, the 1xApollo^® dyeing reaction solution was added to the cells and cultured for 30 min without light. The cell nucleus was stained with Hoechst 33322 for 30 min. The proportion of cells incorporated EdU was determined by fluorescence microscopy (Invitrogen™ EVOS FL Auto 2, Thermo Fisher Scientific, America). Assays were performed in triplicate.