Figure 1.

Enterobactin, chelates intracellular iron in human intestinal epithelia. (a) Iron chelation as indicated by the formation of an orange halo by Ent (0.25 mM to 5 mM) on CAS agar plate over different time periods (1, 6 and 12 h). (b) Iron chelation of FeCl₃ (2.5 mM), Ent (2.5 mM), Ent: FeCl₃ (1:1 ratio) and Ent: FeCl₃ (1:2 ratio) on CAS agar plate over different time periods (1, 6 and 12 h). (c) Line graphs indicate the relative iron chelation activity of Ent (0–50 µM) and Ent: FeCl₃ (1:1 ratio) detected via CAS liquid assay. (d-e) HT29 cells were incubated with 0.5 μM calcein-AM for 15 min and then treated with iron-free or iron-bound Ent (0–25 µM) for 3 h in serum-free media supplemented with 1% penicillin-streptomycin. After washing, iron chelation (LIP = ΔF) was quantitated by flow cytometry. (d) Bar graph represents the iron chelation in HT29 cells after 3 h of iron-free Ent (0–25 µM) treatment. (e) Bar graph shows the iron chelation in HT29 cells after 3 h of Ent (25 µM) and Ent+ Fe³⁺ (1:1 ratio) treatment. (f) HT29 cells (2.0 x10⁶ cells/ml) were treated with Ent (0–25 µM) for 24 h in serum-free media supplemented with 1% penicillin-streptomycin. The release of lactate dehydrogenase (LDH) in the culture supernatant was measured. (g-h) Ent (25 µM)-treated HT29 cells (24 h) were analyzed for cellular apoptosis measured by flow cytometry using Annexin-V/Propidium Iodide positivity. (g) Representative dot plots show the percentage of early and late apoptosis in control and Ent treated HT29 cells. (h) Bar graph indicates the % apoptosis (% Annexin-V+ Propidium Iodide, both early and late apoptosis) at 24 h treatment. Con denotes cells treated with DMSO as vehicle control. In vitro assays were performed in triplicates and data represented as mean ± SEM. * p < .05, ** p < .01, and *** p < .001