Fig. 2: q33 and h33 are protective against the cytocidal effect of Vincristine.

a Vincristine induces HeLa cell death. Cells were incubated for 44 h at 37 °C with 20 nM Vincristine (VCR), then incubated with propidium iodide at 2 μg/mL for 10 min and analyzed by flow cytometry to quantify plasma membrane permeabilization. Histograms show fluorescence intensity detected by flow cytometry (one representative experiment) in the presence (VCR) or Absence (Co.) of Vincristine. % of propidium iodide-positive cells are indicated for both conditions. b Dose–response of Vincristine-induced cell death. HeLa cells were incubated and treated as in (a) with the indicated doses of Vincristine (VCR) and analyzed by flow cytometry. The histogram shows the mean ± SD of 5 independent experiments (***p value < 0.001). c Vincristine induces apoptotic nuclear morphology in HeLa cells. Representative fluorescence microscopy micrographs of cells treated for 44 h with 20 nM vincristine (VCR) or not (Co.) and stained with Hoechst 33342. Vincristine induces progressive nuclear changes with first condensed nuclei (arrows) and then apoptotic bodies (dashed arrow). d Broad-spectrum irreversible caspase inhibitors prevent vincristine-induced cell death. HeLa cells were pretreated for 1 hr with the indicated broad-spectrum irreversible caspases inhibitors (z-VAD(OMe)-fmk at 30 μM, Q-VD-OPh 30 μM, Emricasan 10 μM, or the negative control Q-VE-OPh 30 μM), then treated with vincristine as in panel (a), and analyzed by flow cytometry. Histograms show the mean ± SD of 3 independent experiments (***p value < 0.001). e Effect of the reversible Caspase-2 inhibitors q33 and h33 against vincristine-induced cell death. HeLa cells were pretreated for 1 h with the indicated doses of q33 or h33, then treated with vincristine as in panel a, and analyzed by flow cytometry. Histograms show the mean ± SD of three independent experiments (*p value < 0.1).