Flunarizine hydrochloride

Mibefradil dihydrochloride (Purity: 98.49% by LC-MS) was purchased from MedChem Express (Shanghai, China). Flunarizine hydrochloride and LPS (Escherichia coli 055:B5) were purchased from Sigma-Aldrich (St, Louis, MO, USA). ELISA kits for examination of mouse TNF-α and IL-6 were purchased from Dakewe Biotech Co. Ltd (Beijing, China). Antibodies for phosphorylated p65 and β-actin were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

1 mL cultures of EP1/GFP reporter cells were plated at 100,000 cells/ml and treated with 33 μM of eflornithine (Sigma-Aldrich 1234249), phenothiazine (Sigma-Aldrich P14831), spironolactone (Sigma-Aldrich S3378), or vehicle control and counted daily by hemocytometer. Each day following counting, cells were diluted back to 100,000 cells/ml. For growth curve analysis by flow cytometry, 200 μl cultures of parasites were plated at 100,000 cells/ml and treated with 33 μM of phenothiazine (Sigma-Aldrich P14831), triprolidine hydrochloride (Sigma-Aldrich T6764), flunarizine hydrochloride (Sigma-Aldrich F8257), aprepitant (Sigma-Aldrich SML2215), bufexamac (Sigma-Aldrich B0760), clemastine fumarate salt (Sigma-Aldrich SML0445) or vehicle control. Parasites were stained daily with Sytox Orange and a fixed volume of cells was analyzed by flow cytometry. The number of live cells was calculated as the number of Sytox Orange negative cells falling within the live gate. Each day following flow analysis, cells were diluted back to 100,000 cells/ml according to the number of live cells counted.