Amphotericin

S. mansoni is maintained in the laboratory using the intermediate snail host Biomphalaria glabrata and as definitive host the golden hamster (Mesocricetus auratus). Cercariae were released from infected snails and mechanically transformed to obtain schistosomula in vitro [22 (link)]. Newly transformed schistosomula were maintained for 12 h in M169 (Vitrocell) medium supplemented with 2% fetal bovine serum (FBS) (Vitrocell), 1 μM serotonin, 0.5 μM hypoxanthine, 1 μM hydrocortisone, 0.2 μM triiodothyronine, penicillin/streptomycin, amphotericin, gentamicin (Vitrocell) at 37°C and 5% CO₂ [23 (link)], after which time the drug treatment was initiated, as described below. Adult worms were obtained from 7-week infected hamsters by left ventricular perfusion, and release of worms from the hepatic portal vein. Paired worms were maintained in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Vitrocell), penicillin/streptomycin, amphotericin (Vitrocell) at 37°C and 5% CO₂.
The parasites were treated with 1 μM Trichostatin A (Cayman Chemical), a concentration that has been shown by Dubois et al. [15 (link)] to be sub-lethal, and the negative controls with an equivalent amount of ethanol (vehicle of TSA), for 12, 24 and 48 h for microarray experiments, and for 12 h for ChIP-qPCR and western-blotting experiments.

Schistosomula or adult worms were treated with different concentrations of LSD1 inhibitors, as indicated in the figure legends. For each treatment condition, 10 worm pairs were maintained in 60-mm diameter culture dishes in 2 mL of culture medium (medium M169 (Gibco) supplemented with 10% fetal bovine serum (Vitrocell), penicillin/streptomycin, amphotericin and gentamicin (Vitrocell). Schistosomula were maintained in 96-well or 24-well culture plates, depending on the experiment, with 200 μL or 1 mL of culture medium M169 (Gibco), respectively, supplemented with 2% fetal bovine serum (Vitrocell), 1 μM serotonin, 0.5 μM hypoxanthine, 1 μM hydrocortisone, 0.2 μM triiodothyronine, penicillin/streptomycin, amphotericin and gentamycin (Vitrocell). Parasites were maintained at 37°C in 5% CO₂ with a humid atmosphere. The medium containing the LSD1 inhibitors or DMSO (vehicle) was refreshed every 24 h during the treatment period (1–4 days).

Newly transformed schistosomula (NTS) were maintained for 2 h in M169 (Vitrocell) medium supplemented with 2% fetal bovine serum (FBS) (Vitrocell), 1 μM serotonin, 0.5 μM hypoxanthine,1 μM hydrocortisone, 0.2 μM triiodothyronine, penicillin/streptomycin, amphotericin, gentamicin (Vitrocell) at 37°C and 5% CO₂ [28 (link)]. Only after 2 h incubation in culture medium the NJ series treatment was initiated as follows: 6.25 μM, 12.5 μM, 25 μM or 50 μM compound, for 1–5 days. Paired adult worms freshly perfused from infected hamsters (see above) were maintained in culture in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Vitrocell), penicillin/streptomycin, amphotericin, gentamicin (Vitrocell) at 37°C and 5% CO₂ for 2 h prior to the beginning of treatment with NJ series compound as follows: 12.5 μM, 25 μM or 50 μM compound, for 1–3 days of treatment. In all cases, NJ series compounds were prepared from a stock solution of 20 mM in dimethyl sulfoxide (DMSO), and the equivalent amount of DMSO was added to the control assays.