Colchicine

The following reagents were used: CBD (Sigma, United States); colchicine (Beyotime, China); CCl₄ (Aladdin, China); peanut oil (Yuanye, China); ELISA kits (Novus, United States); hematoxylin and eosin (HE) and Masson assay kits (Beyotime, China); aspartate aminotransferase (AST) and hyaluronic acid (HA) kits (Nanjing Jiancheng Bioengineering Institute, China); RIPA lysis buffer and a BCA kit (Solaibao, China); COX-2, p-IκBα, and IκBα antibodies (Wanleibio, China); p-NF-κB, NF-κB, p-p38 MAPK, and p-38 MAPK antibodies (CST, United States); TGF-β1, α-SMA, COL-I, PPAR-α and GAPDH antibodies (Abcam, United States); horseradish peroxidase-labeled secondary antibodies (Bioprimacy, China); chemiluminescence (ECL) color developing solution (Merck, United States); an RNeasy mini kit (Axygen, United States); a PrimeScript RT reagent kit with gDNA Eraser and SYBR Green Master Mix (Takara, Japan); an automatic chemical analyzer (Hitachi, Japan); and light microscopy (Nikon, Japan).

The MIR503HG knockout hES‐H9 cells were cultured until reaching 80% confluency. Colchicine (ST1173, Beyotime) was added to the medium and incubated for 3 h at 37°C, and then cells were digested into single cell by Accutase (07920, STEMCELL Technologies). The cells were resuspended in 75 mm KCl solution for 30 min and then incubated in a fixative containing ethanol/acetic acid (3:1, v/v) for 20 min at RT. 20 µl cells suspension were applied to each clean slide. Dyed by Giemsa stain (C0131, Beyotime) for 30 min at RT, 20 metaphases were counted for each sample, and then chromosome analysis were performed using the Leica DMRB epifluorescence microscope.

After 0.1 μg/ml colchicine (Beyotime, SC7992‐10 mM) incubation at 37°C for 3 h, cell cycle was arrested in metaphase as expected. All cells were collected by gently pipetting, washed once with 1 × PBS and incubated with 0.075 M KCl at 37°C for 30 min. After incubation, cells were fixed with 3:1 methanol/glacial acid and 0.075 M KCl (1:4 fixative/0.075 M KCl) for 10 min. The fixation process was repeated for three times. Then, cells were dropped onto a wet glass slide and dried in a 75°C oven. After 5 min rehydration with PBS, all slides were fixed with chromosome diffusion +2% paraformaldehyde for 10 min. Next, slides were treated with pepsin (#P8390; Solarbio) at 37°C and then washed with PBS twice, 5 min each round. Then, the slides were sequentially treated with 70%, 85% and 100% ethanol, 2 min each step, and dried for at least 30 min. Next, 200 μl hybridization buffer (2% BSA 10 μl, 0.8 μl final concentration 125 nmol Telc‐FITC (#200616PL‐01; panagene), 0.6 × SSC 20 μl, deionized formamide 140 μl, add ddH₂O 56.4 µl) was added on slides. Before staining, slides were heated at 85°C for 3 min for DNA denaturing and then incubated at 37°C for 2 h. After washing with washing buffer I (10 mM Tris, 70% formamide, 2 × 15 min) and 0.1% PBST (3 × 5 min), nucleus was stained with DAPI, and slides were mounted with glycerol and imaged by fluorescent confocal microscopy.