I bet151

TH1834 (Axon, cat#2339) or I‐BET151 (Selleckchem) were dissolved in DMSO and used for cell treatment studies. To perform treatment studies with the leukemia-bearing mice (usually seven days post-BMT), I‐BET151 was first dissolved in DMSO to make a stock solution of 120 mg/mL, followed by dilution to 6 mg/mL by mixing 50uL of the I‐BET151 stock with 950uL of vehicle solution (20% Kolliphor HS 15 [v/v] in 0.9% of NaCl; Sigma, 42966). I‐BET151 or mock vehicle control was delivered via intraperitoneal (i.p.) injection at a daily dose of 30 mg/kg body weight for three weeks (5 days’ injections per week).

The list of pharmacologically active compounds (LOPAC^®) library contains 1280 experimentally validated small molecules that belong to diverse chemical classes. The ENZO^® SCREEN-WELL^® FDA-approved drug library V2 contains 774 FDA drug compounds belonging to various indication classes. LOPAC^® and ENZO^® compound libraries were kindly provided by Fraunhofer ITMP in Hamburg. Screening compounds were maintained in DMSO at 10 mM and stored on 384-well plates (Echo Qualified 384-well plates, Labcyte, San Jose, CA, USA) at −20 °C. The composition of the LOPAC^® and ENZO^® libraries is shown in supplementary Tables S1 and S2, respectively.
I-BET151, Ro 11-1464, trichostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA) were purchased from Sigma Aldrich (St. Louis, MO, USA). Stock solutions of I-BET151 (10 mM), Ro 11-1464 (10 mM), TSA (6.6 mM), and SAHA (10 mM) were prepared in DMSO (Sigma Aldrich) and stored at −20 °C. Each of the conditioned media was freshly prepared before treatment.

A total of 1,585 drugs were tested from the Johns Hopkins University Clinical Compound Library, which had patented compounds removed prior to drug screening. Drug screening was conducted with the EP1/GFP parasites plated with 1,000 cells in 100 μL cultures and 33 μM of drug. After 3 days of growth, cells were screened for GFP fluorescence via flow cytometry. To test for autofluorescence of candidate hits, 300,000 cells/100μL culture were plated with 33 μM of each drug and immediately screened for fluorescence via flow cytometry. Each plate also contained three wells of negative control cells treated with DMSO and a well of positive control cells treated with 20μM I-BET151 (Sigma-Aldrich).
All samples with an average median GFP fluorescence intensity that was 1.5-fold or higher than the +DMSO control from the first two rounds of screening were selected for further screening. Drugs tested in triplicate were subjected to a two-sided, unpaired Student’s t-test. To identify drugs that inhibit trypanosome growth or those that are trypanocidal, we used flow cytometry to calculate total cell counts/10μl of volume. Control cells typically showed ~35000 cells/10μl volume within the live gate, so we identified trypanocidal drugs as those that resulted in counts between 350–3500, 35–350, and <35 in the live gate.