Fig. 6.

Comparison of PAPTP and Venetoclax effects in human B CLL cells ex vivo. a-b Mitochondrial ROS production (a) and mitochondrial membrane potential (b) were measured, respectively, by MITOSOX (a) and TMRM (b) fluorescence from ex vivo CLL B cells, isolated from 3 CLL patients, either treated with 3 nM Venetoclax or untreated. The percentage of the initial values of MITOSOX and TMRM signals are reported as a function of treatment time and are shown as mean ± SD. For comparison of the kinetics induced by Venetoclax and PAPTP, data shown in Fig. 1b for PAPTP are indicated also here. Statistical significance of differences was assessed with Multiple unpaired t-test. c Cytochrome c levels in mitochondria and cytoplasm were evaluated by western blotting from ex vivo human CLL B cells treated with DMSO or 3 nM Venetoclax (25 μg protein/lane). ATP5A and β-actin were chosen as mitochondrial and cytoplasmic markers and are shown as well. d, e Ex vivo peripheral blood cells, isolated from 7 CLL patients were treated with 0.2 μM or 2 μM PAPTP alone (d) or in combination with 2 nM Venetoclax (e). The percentage of CD19⁺CD5⁺ dead cells, estimated with Annexin V staining, was assessed by flow cytometry for each condition and is shown as fold induction of the untreated control (alone) and mean ± SEM values are depicted by histograms. Statistical significance of differences was assessed with the Wilcoxon test. f Percentage of dead cells, based on Annexin V staining, was assessed by flow cytometry on cultured ex vivo B CLL cells isolated from ibrutinib-resistant patients. Cells were treated with different concentrations of TRAM-34 or with different concentrations of PAPTP, alone or in combination, as indicated. Data were obtained from 4 patients and are represented as mean ± SEM. Statistical significance of group differences compared to the untreated one were assessed with the Kruskal-Wallis test (*** p ≤ 0.001); statistical significance of differences between individual groups were assessed with the Mann–Whitney test