Fig. 4.

Caspase cascade activation after Amblyomin-X treatment in tumor cells. a Cells were pre-incubated for 2 h with ZVAD-FMK (50 µM) or DEVD-CHO (10 µM) followed by incubation with Amblyomin-X (1 µM) for 48 h at 37 °C. b Caspase activity was measured using CellEvent™ Caspase-3/7 Green Detection Reagent (Molecular Probes), according to manufacturer’s instructions. Cells treated with vehicle (PBS), or 0.5 µM of Amblyomin-X for 48 h, or with MG-132 (2.5 µM) and TAPS (1 µM) for 24 h each. Then, cells were stained with CellEvent™ Caspase-3/7 Green Detection Reagent and were analyzed by flow cytometer. c Bars graph (fluorescence intensity) values ± SD obtained from B (three independent experiments). d After the period of treatment, cells were lysed with RIPA buffer and 30 µg of protein extract was separated on SDS-PAGE. Western blot analysis were performed using anti-PARP and anti-GAPDH (endogenous control). *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 (Ctrl vs Amblyomin-X or Amblyomin-X vs inhibitors)