Intestinal IL-17A⁺ total ILC3s, IL-17A⁺ DN ILC3s (e), IL-22⁺ total ILC3s and IL-22⁺ DN ILC3s (f) quantified in Atg5^fl/fl and Rorc^creAtg5^fl/fl mice.

Experimental Design Core Objective: Flow cytometry was performed to evaluate the effects of Atg5-mediated autophagy deficiency and metabolic interventions (lactate supplementation, etomoxir treatment) on the effector function of total group 3 innate lymphoid cells (ILC3s) and double-negative (DN) ILC3 subsets in the intestinal lamina propria of mice with necrotizing enterocolitis (NEC). Specifically, we quantified the percentage of IL-17A-producing and IL-22-producing cells within total ILC3s and DN ILC3 subsets across all experimental groups. Animal Model:Genetically modified mice on a C57BL/6 background were used in this study: Atg5fl/fl mice: Wild-type control mice with intact autophagy function in RORγt+ cells RorccreAtg5fl/fl mice: RORγt+ cell-specific conditional Atg5 knockout mice, with autophagy deficiency specifically restricted to the ILC3 lineageAll mice were subjected to standardized experimental NEC induction prior to intervention and sample collection. Experimental Groups (4 groups total): Atg5fl/fl + DMSO: Vehicle control group (autophagy-intact ILC3s, vehicle treatment) RorccreAtg5fl/fl + DMSO: Autophagy-deficient vehicle group (autophagy-deficient ILC3s, vehicle treatment) RorccreAtg5fl/fl + Lactate: Metabolic intervention group (autophagy-deficient ILC3s, lactate supplementation) RorccreAtg5fl/fl + Etomoxir: Metabolic intervention group (autophagy-deficient ILC3s, fatty acid oxidation inhibitor treatment) Detection Targets (quantified for both total ILC3s and DN ILC3 subsets): Percentage of IL-17A+ cells within ILC3s/ILC3 subsets Percentage of IL-22+ cells within ILC3s/ILC3 subsets Biological Replicates: 6 biological replicates (individual mice) per group Statistical Analysis: One-way ANOVA with Tukey’s post-hoc test was used for multiple group comparisons. Statistical significance was defined as P < 0.05. Flow cytometry data were acquired using a CytoFLEX S flow cytometer (Beckman Coulter). The following fluorochrome-conjugated antibodies were used for cell surface and intranuclear staining in this study:NKp46-FITC, Lin-APC-Cy7, live/dead-BV510, CD4-Pacific Blue (PB), CD90.2-PE-Cy7, IL17A/IL22-PE, CCR6-APC, and RORγt-PerCP-Cy5.5. Partial data were excluded from the repository due to database size constraints (not omitted); all underlying data are available upon reasonable request.