Figure 5.

Ent treatment dampens Lcn2 secretion and host Lcn2 also impedes iron chelation property of Ent. HT29, DLD-1, and Caco-2/BBe 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. (a) Bar graphs represent the iron chelation in HT29, DLD-1, and Caco-2/BBe cells after 3 h of iron-free Ent (0–25 µM) treatment. (b) Histograms represent the flow cytometric analysis of intracellular iron chelation in control (vehicle treatment, red) and Ent treated intestinal epithelial cells (blue). (c-d) HT29, DLD-1 and Caco-2/BBe cell monolayers were incubated with Ent (0–25 µM) for 24 h and culture supernatants were collected and assessed for (c) extracellular Lcn2 secretion and (d) intracellular Lcn2 by ELISA and normalized with the protein concentration. (e) The monolayers of HT29 and Caco-2/BBe cells were incubated overnight with Ent (25 µM), FeCl₃ (Fe³⁺) or Ent with an equimolar concentration of FeCl₃ and supernatants were analyzed for Lcn2. (f) HT29 monolayers were pre-incubated with either (i) Ent (25 µM, 1 h) or (ii) rec-Lcn2 (0.625 µM, 1 h) and then treated with either rec-Lcn2 or Ent or Ent+ rec-Lcn2 complex for 24 h in serum-free media supplemented with 1% penicillin-streptomycin in subsequent wells and supernatants were assayed for IL-8 via ELISA. (g) HT29 monolayers were pre-incubated with rec-Lcn2 (0.625 and 1.25 µM, 1 h) and then treated with Ent (25 µM) for 24 h in serum-free media supplemented with 1% penicillin-streptomycin in subsequent wells and supernatants were examined for IL-8 via ELISA. In vitro assays were performed in triplicates and data represented as mean ± SEM. * p < .05, **p < .01,*** p < .001 and **** p < .0001