Chlorophenol red β d galactopyranoside

The indicated B3Z clones were photoactivated using 470 nm LEDs for 10 s every 5 min for a total period of 1 h. After three additional hours of culture, cells were washed twice in phosphate-buffered saline (PBS) and lysed in 100 μL per well of CPRG buffer (PBS + 9 mM MgCl2 + 0.125% NP40 + 100 μm β-mercaptoethanol + 0.15 mM chlorophenol red- β-D-galactopyranoside (Roche, #10884308001)). Plates were incubated in the dark at room temperature for 30 min to 1 h and the optical density was read at 570 nm (reading at 620 nm was used as reference and subtracted).

The evaluation of the antiprotozoal activity against trypanosomes was carried out using the recombinant strain Tulahuen clone C4 of T. cruzi trypomastigotes (ATCC, Manassas, VA, USA). This strain expresses the β-galactosidase enzyme used as a reporter to assess the viability of the trypanosomatid [41 (link)]. The parasites were maintained at 37 °C under a 5% CO₂ atmosphere in RPMI-1640 culture medium with L-glutamine, 4-(2-Hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES) buffer, NaHCO₃, 10% FBS, and 0.05% gentamicin (50 mg/mL) as supplements. Vero cells were cultured for 24 h, and one day prior to the experiment, the cells were infected with trypomastigotes. After additional 24 h of infection, bufadienolides were dissolved in DMSO and tested at four different concentrations by duplicate for five days. Benznidazole was used as the positive control. To determine antitrypanosomal activity, 25 µL of chlorophenol-red-β-D-galactopyranoside (Roche Applied Science) (900 µM) were added to each well and allowed to react with the β-galactosidase of the remaining living parasites for 4.5 h. Absorbance was calculated at 570 nm using a microtiter plate reader (Sinergy HT, BioTek Instruments Inc, Winooski, VT, USA).

For reporter gene assays, HEK293 cells were seeded into 24-well plates at a density of 0.5–1 × 10⁵ cells/well. The next day, DNA plasmids, including ERK5 (WT or mutant) and reporter genes (total 1 μg/well), were combined with Lipofectamine 2000 (1 μl/well) and mixed gently in serum-free DMEM (50 μl) and added to the culture plates for 4–6 h. Cells were then serum starved overnight and incubated with EGF in the presence or absence of U0126 at 37°C for 6 h. Cells were lysed in lysis buffer (1% Triton X-100, 110 mM K₂HPO₄, 15 mM KH₂PO₄, pH 7.8) (100 μl/well) and centrifuged to remove cell debris. The resulting supernatant (50 μl/tube) was mixed with 300 μl assay buffer (25 mM Gly-Gly, 15 mM MgSO₄, 5 mM ATP, 10 mM NaOH). The luciferase reaction was started by the addition of 100 μl luciferin solution (150 μM), and luciferase activity was measured using a luminometer (LB9575; Berthold, Bad Wildbad, Germany). As an internal control, β-actin promoter-driven β-galactosidase activity was measured to normalize for transfection efficiency. The cell lysate (20 μl) was incubated with 100 μl reaction buffer (100 mM Hepes, 150 mM NaCl, 2 mM MgCl₂, pH 7.0) supplemented with chlorophenol red-β-D-galactopyranoside (2 mg/ml) (Roche, Indianapolis, IN), and the absorbance at 595 nm was measured.