Colchicine

To evaluate the effects of protein palmitoylation inhibitor 2‐BP (Sigma‐Aldrich, cat# 21604) on ookinete differentiation, 2‐BP was added to in vitro ookinete cultures with final concentration of 100 μM 1 h after fertilization. To evaluate the effects of microtubule assembly inhibitor colchicine (MedChemExpress, cat# HY‐16569) on ookinete differentiation, colchicine was added to cultures with final concentration of 100 μM 1 h after fertilization. Ookinete cultures were harvested at different times for further analysis.

ASIC1a (1:1,000 dilution, Bioss), Collagen-1 (1:1,000 dilution, Proteintech), α-SMA (1:1,000 dilution, Bioss), β-Actin (1:1,000 dilution, Proteintech), CaM (1:1,000 dilution, Affinity), CaMK II (1:1,000 dilution, Affinity), NFAT (1:1,000 dilution, Affinity), MMP-13 (1:1,000 dilution, Bimake.cn), NF-κB (1:1,000 dilution, Bimake.cn), KN93, and colchicine were purchased from MedChemExpress (MCE, United States). Spider-venompeptide (PcTx-1) was purchased from Abcam (Cambridge, United States). High-sugar DMEM culture medium and phosphate buffered saline (PBS) were purchased from HyClone (United States). Horseradish enzyme labeled anti-rabbit IgG and horseradish enzyme labeled anti-mouse IgG were purchased from Bioss (Beijing, China). PVDF membrane was purchased from Millipore (United States). Fluo-3AM calcium fluorescent probe was purchased from beyotime (Shanghai, China).Lipofectamine 2000, TRIzol Reagent, and Opti-MEM were purchased from Invitrogen (Invitrogen, United States). Amiloride was purchased from Sigma-Aldrich (St. Louis, MO, United States). Rapid Gel Preparation Kits were purchased from EpiZyme (EpiZyme, China). Cell cycle detection kit and CCK-8 cell proliferation toxicity assay kit were purchased from BestBio (Beijing, China).

Karyotype of cells was identified as described previously [49 (link)]. Briefly, cells were incubated with colchicine (Medchem Express LLC.) for 2 h at a confluency of 90%. Accutase™ treated cells were then incubated with KCl containing hypotonic solution for 20 min. Cells were fixed with acetic acid/methanol (3:1) overnight at 4 °C, and subsequently stained and photographed at the department of medical genetics, PKU School of Basic Medical Sciences (Beijing, China).