R elated to Fig. 3 . (A) Volcano plot of bulk mRNA sequencing data illustrating GC-enriched genes differentially expressed between ConvR and GF adult colon. (B) Reactome pathways significantly enriched in microbiota-regulated GC genes identified as positively correlated with postnatal age. (C) Confocal micrographs of fixed colonic tissue from adult ConvR and GF mice stained for DNA (grey) and Apo-Muc2 (red). (D) Quantification of mCherry⁺ GCs from colonic whole mounts based on images shown in Fig. 3 J. P3 samples are divided into late epithelium (LE) and early epithelium (EE). (E) Quantification of total epithelial cells/crypt in P1–9 neonatal and adult ConvR and GF tissues based on images shown in Fig. 3 G and Fig. S1 C. (F) Quantification of Apo-Muc2–positive cells as a fraction of total epithelial cells in adult GF and conventionalized (ConvD) mice at different time points after colonization. Data represent n = 4 (A–D) or n = 3 (D) animals per group. All error-bar graphs show median and interquartile range. All data are pooled from at least two independent litters or experiments. Statistical comparisons by one-way ANOVA and Fisher’s LSD; P < 0.05 (*), P < 0.01 (**). Image scale bars are 50 µm.