Optimem medium

FTC-133 cells were used to study the effect of PROX1 knock-down on hallmark characteristics of malignant cells: migration, invasion, motility, proliferation, apoptosis and anchorage–independent growth. Cells were grown to 60-70% confluence in 24-well or 6-well plates and transiently transfected with either non-targeted pool of small interfering RNAs (MISSION^® siRNA Universal Negative Control #1, Sigma-Aldrich, St. Louis, MO, USA), a pool of specific siRNAs against human PROX1 (MISSION^® esiRNA, Sigma-Aldrich, EHU053851) or with PROX1 siRNA (h) (sc-106451, Santa Cruz Biotechnology, USA), using Lipofectamine^® 2000 Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturers’ protocol. The optimal transfection efficiency was obtained using a cell density of 4.5x10³ per well, 30 nM siRNAs in OptiMem medium (Roche, Basel, Switzerland), and 1.5 μl Lipofectamine/OptiMem. Cells were incubated with the transfection mix for 48 hours and then harvested for RNA purification or used for other assays. The experiments were conducted on at least three different cell passages and run in triplicate (three samples of the same cell passage).

Trophozoites were seeded into 96-well plates at 0.5 × 10⁴ cells/well in 280 µL of BI-S-33 medium containing 5–80 µM Miltefosine. After 18 h of culture, the medium was removed and the viability of attached trophozoites was estimated by measuring of the absorbance at 450 nm using a DTX880 Multimode Detector (Beckman Coulter, Brea, CA, USA) after incubating of the trophozoites with 100 µL of Opti-MEM medium containing 10% WST-1 reagent (Roche, Basel, Switzerland) for 20 min at 37 °C [63 (link)]. Experiments were repeated three times with triplicate samples evaluated in each experiment.