Fig. 3: PAMT-001-mediated mitochondrial dysfunction and mtROS activate the intrinsic apoptotic process.

A GSEA against GOBP_Positive regulation of intrinsic apoptosis. Genes were ranked based on fold changes between vehicle and PAMT-001. B, C Flow cytometry showing apoptosis of Annexin V/PI-stained primary AML cells #104 (B). Bar plot showing fractions (%) of Annexin V-positive cells (C). P-values were calculated using one-way ANOVA for multiple comparisons (mean ± SD, n = 3). Representative transmission electron microscopy image of apoptotic blebbing (yellow arrowhead) in PAMT-001-treated HL-60 cells (2.5 µM, 12 h). E Western blotting of apoptosis-associated proteins in PAMT-001 treatment. Time-dependent cleavage of caspase-9 and -3 by PAMT-001 in AML cell lines (KG1α (left), HL-60 (middle), and THP-1 (right). F Western blotting of cytochrome C released from mitochondria into the cytoplasm in PAMT-001-treated KG1α cells. G LD₅₀ value of PAMT-001 with and without Q-VD-OPh in KG1α analyzed using MTT assay. LD₅₀ values were calculated through [inhibitor] vs. normalized response—Variable slope in Prism 8. P-values were calculated using the extra sum of squares F-test (mean ± SD, n = 4). H Representative flow cytometry of mtROS levels in primary AML cells (#99) treated with PAMT-001 (5 μM, 18 h), as detected via MitoSOX staining. The bar plot shows fractions (%) of MitoSOX-positive cells. P-values were calculated using a two-tailed t-test (mean ± SD, n = 3). I, J Cell death measured using flow cytometry with Annexin V/PI staining of KG1α cells. Cells were cotreated with N-acetylcysteine (NAC) and PAMT-001 (3.75 μM) for 18 h (I). Bar plot shows fractions (%) of Annexin V-positive cells (J). P-values were calculated using one-way ANOVA for multiple comparisons (mean ± SD, n = 3). K Cell death was measured using the tryptophan exclusion assay of K562-luc cells. PAMT-001-induced cell death was partially rescued by MitoQ. P-values were calculated using a two-tailed t-test (mean ± SD, n = 3).