Fig. 2: THZ-P1-2 induces apoptosis and dysfunction in mitochondria and autophagic flux.
A Apoptosis was detected by flow cytometry in MV4-11, OCI-AML3, Jurkat, and NALM6 cells treated with vehicle or with increasing concentrations of THZ-P1-2 (1.6, 3.2, and 6.4 μM) for 24 h using an APC-annexin V/PI staining method. Representative dot plots are shown for each condition. The upper and lower right quadrants (Q2 plus Q3) cumulatively contain the apoptotic cell population (annexin V+ cells). Bar graphs represent the mean ± SD of at least three independent experiments. The p values and cell lines are indicated in the graphs; *p < 0.05, **p < 0.01, ***p < 0.0001; ANOVA and Bonferroni post-test. B Mitochondrial membrane potential (ΔΨM) analysis was evaluated using the JC-1 staining method and flow cytometry. Leukemic cells were treated with vehicle or THZ-P1-2 (1.6, 3.2, and 6.4 μM) for 24 h. Representative dot plots are shown for each condition; the gate FL-2 contains cells with intact mitochondria and the gate FL-2/FL-1 contains cells with damaged mitochondria. Bar graphs represent the mean ± SD of at least three independent experiments and the p values are indicated; **p < 0.01, ***p < 0.0001; ANOVA and Bonferroni post-test. C The evaluation of acidic vesicular organelles (AVOs) was investigated through acridine orange (AO) labeling and flow cytometry in AML and ALL cell lines treated with vehicle or THZ-P1-2 (1.6, 3.2, and 6.4 μM) for 24 h. Bar graphs represent the mean ± SD of at least three independent experiments and the p values are indicated; *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA and Bonferroni post-test. D Alternatively, the presence of AVOs was confirmed by immunofluorescence on OCI-AML3 and NALM6 cell lines treated with vehicle or THZ-P1-2 (3.2 and 6.4 μM) for 72 h on a Lionheart FX automated microscope at magnification, ×400. The overlapping GFP and RFP channels are shown.
