Melatonin effects are associated with the lamina propria Th17/Treg balance. (A) Representative flow cytometry plots of RORγt and Foxp3 expression in gated lamina propria CD4+ T cells from ileum sections of breastfed (BF) and necrotizing enterocolitis (NEC) model pups upon melatonin treatment (MEL) or treatment with vehicle (VEH). (B, C) Quantification of the percentages of Th17 (B) and Treg (C) cells in (A), n = 5 per group. (D) Immunoblot analysis of RORγt, Foxp3, p-STAT3, STAT3, p-STAT5, and STAT5 in the ilea of BF, VEH, and MEL pups; β-actin was used as an internal control. (E-H) Quantification of RORγt (E), Foxp3 (F), p-STAT3/STAT3 (G), and p-STAT5/STAT5 (H), n = 6 per group. Each symbol (B, C, E-H) represents an individual mouse; column graphs represent the mean with error bars indicating standard deviation (SD), *P < 0.05; ***P < 0.001; ns: not significant. P values were derived through one-way ANOVA followed by the Bonferroni multiple comparison test (B, C, E-H). Data are representative of three independent experiments (B, C, E-H).