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. 2025 Mar 28;23(4):146. doi: 10.3390/md23040146

Figure 2.

Figure 2

The biochemical inactivation and cellular depletion of heme upon XanDME binding. (A) Dose-dependent inhibition of the GSH-mediated degradation of hemin (25 μM) by XanDME. (B) In vitro peroxidase activity assay conducted in the presence of hemin, with or without XanDME. Hemin (5 nM) was preincubated with either XanDME (25 μM) (blue) or assay buffer (purple). The time dependent response of the hemin-promoted peroxidase activity was represented by the absorption (λem = 570 nm) in kinetic mode. (C) Flow cytometry measurements of the intracellular XanDME in TNBC cells (MDA-MB-468, MDA-MB-231, 4T1) post 24 h treatment at 5, 10, 20 μM using the PB450 channel. The data represent the average values ± SD of independent experiments (n = 3 per group). (D) The regulatory heme (RH) content in 4T1 cells measured by hemin colorimetric assay post 48 h treatment with XanDME (5 μM). (E) The RH content in MDA-MB-231 cells by hemin colorimetric assay. Cells were treated with XanDME (5 μM) for 48 h, with or without a 2 h pretreatment of hemin (10 μM). (F) The total heme content in MDA-MB-231 cells measured by fluorescence (λem = 610 nm, 660 nm) post 24 h treatment with XanDME (5 μM). (G) Porphyrin (PPIX) content in cell lysate measured by fluorescence (λex = 406 nm). MDA-MB-231 cells were incubated with either XanDME (5 µM) or DMSO for 24 h, following a 2 h pretreatment with ALA (0.1 mM) (** p < 0.01; *** p < 0.001).