*3.7. Cytotoxicity Assay*

The long-term human microvascular endothelial cell line (HMEC-1) was maintained in MCDB 131 medium (Invitrogen, Milan, Italy) supplemented with 10% fetal calf serum (HyClone, Celbio, Milan, Italy), 10 ng/mL of epidermal growth factor,1 μg/ml of hydrocortisone, 2 mM glutamine and 20 mM Hepes buffer (EuroClone). For the cytotoxicity assays, HMEC-1 were plated at 10<sup>5</sup> cells/mL in 96-well flat bottom microplates. THP-1 cells were plated at 5 × 10<sup>5</sup> cells/mL in 96-well flat bottom microplates and treated with 0.1 μM PMA for 48 h to achieve differentiation into macrophages. Cells were then treated with serial dilutions of test compounds and cell proliferation evaluated using the MTT assay described for promastigotes. The results are expressed as IC50, which is the dose of compound necessary to inhibit cell growth by 50%.

### *3.8. In vitro E*ff*ects of Compounds on S. mansoni Parasites and Eggs*

Schistosomula were prepared by mechanical transformation of cercariae, as previously described [56]. The ATP-based viability assay with CellTiterGlo (Promega, Italy) on the larval stage of schistosomes was carried out in 96-well, black, tissue culture plates by adaptation of a protocol previously set up in our laboratory [56]. DMSO (vehicle) and gambogic acid (10 μM) were used as the negative and positive controls in each plate and the percentage of viability for each compound was calculated as the ATP reduction against vehicle (0%) and gambogic acid (100%).

All Animal work was approved by the National Research Council, Institute of Cell Biology and Neurobiology animal welfare committee (OPBA) and by the competent authorities of the Italian Ministry of Health, DGSAF, Rome (authorization no. 25/2014-PR and no. 336/2018-PR). All experiments were conducted in respect to the 3R rules according to the ethical and safety rules and guidelines for the use of animals in biomedical research provided by the relevant Italian law and European Union Directive (Italian Legislative Decree 26/2014 and 2010/63/EU) and the International Guiding Principles for Biomedical Research involving animals (Council for the International Organizations of Medical Sciences, Geneva, Switzerland).

A Puerto Rican strain of *S. mansoni* was maintained by passage through albino *Biomphalaria glabrata*, as the intermediate host, and ICR (CD-1) outbred female mice as previously described [56]. Female 4 to 7-week-old mice (Envigo, Udine, Italy) were infected with 200–400 double sex *S. mansoni* cercariae by the tail immersion technique. Adult pairs were harvested from mice 7–8 weeks after infection by reversed perfusion of the hepatic portal system and mesenteric veins. For the viability assays, 5 couples were incubated with the compounds in 3 mL DMEM complete tissue culture medium containing 10% FBS for up to 7 days and phenotypic score was assigned as previously described [57]. For egg-treatment, 5 worm pairs were incubated for 48 h in 3 mL complete tissue culture medium; next the parasites were removed and vehicle (DMSO) or compounds **1**–**3** were added to each plate containing the eggs previously laid in vitro and observed for 72 h as previously reported [14]. Briefly, images were recorded with a BX41 Olympus microscope and a bright field objective 10× served by a SPOT RT 220-3 Diagnostic Instrument Inc camera. Egg maturation/morphological score was assigned based on the Vogel and Prata' staging system of egg maturation [47].
