SIRT1 pathway inhibition attenuates maintenance of the Th17/Treg balance. (A) Representative flow cytometric plots of IL-17 and Foxp3 expression in gated lamina propria CD4+ T cells from ileum sections of pups following necrotizing enterocolitis (NEC) induction upon treatment with vehicle (VEH) alone or with melatonin (MEL) or melatonin combined with Ex-527 (MEL + Ex-527). (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 ilea of VEH, MEL, and MEL + Ex-527 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 from one-way ANOVA followed by a Bonferroni multiple comparison test (B, C, E-H). Data are representative of three independent experiments (B, C, E-H).