Mouse small intestinal crypt cells express PCNA.

<p>PCNA expression was analyzed by immunoblotting. 50 µg total proteins from extracted mouse small intestinal crypt cells and cultured rat intestinal crypt cell line IEC-6 were loaded. PCNA was expressed in both cell types and with significantly higher abundance in mouse small intestinal crypt cells (<i>P</i><0.01) than in IEC-6 cells. β-actin served as a loading control. Values are means ± SEM run in triplicates.</p

IGF-1 stimulates mouse small intestinal crypt cell proliferation.

<p>Crypt cells in 96-well plate were treated with 20 ng/ml IGF-1; the control group was grown in the crypt cell growth medium alone. After treatment for 24 h, rate of cell proliferation was measured by Brdu incorporation assay. IGF-1 significantly stimulated proliferation of the crypt cells (<i>P</i><0.05). Values are means ± SEM run in triplicates; asterisks indicate significant differences between IGF-1 treatment and control groups.</p

CCNE1, CDK2, and CREB1 are directly involved in IGF-1 stimulated crypt cell proliferation.

<p>(A) immunoblotting analyses of CCNE1, CDK2, and CREB1 protein in mouse small intestinal crypt cells with or without IGF-1 treatment for 24 h. Data were normalized to β-actin. Values are means ± SEM run in triplicates, asterisks indicate significant differences between control and IGF-1 treated cells. (B) CCNE1, CDK2, and CREB1 proteins were knocked-down by siRNA in IEC-6 cells before treated with IGF-1. Cell proliferation was measured 24 h after IGF-1 treatment. Values are means ± SEM run in triplicates, letters indicate significant differences between time points.</p

CCNE1, CDK2, and CREB1 are direct targets of miR-103 in mouse small intestinal crypt cells during IGF-1 stimulation.

<p>(A) sequence alignment between miR-103 seed region and the seed matches on CCNE1, CDK2, and CREB1 mRNA 3′UTR region. The analyses were performed using the miRBase target database. The line indicates conserved seed match (A–T, C–G) in human and mouse, whereas the dot indicates seed match in one of the two species. (B, C) miR-103 directly binds and represses CCNE1, CDK2, and CREB1 mRNA through 3′UTR. pGL3-control luciferase vectors containing the mRNA 3′UTR of respective genes of human (B) or mouse (C) origin were co-transfected with the indicated amount of miR-103 mimic in HEK293 cells, and luciferase activity was analyzed 24 h post-transfection. The structurally unrelated miRNA cel-miR-67 served as negtive control. (D) CCNE1, CDK2, and CREB1 protein expression were examined in IEC-6 cells after transfection with 50 nM miR-103 inhibitor. Scramble (scr) miRNA transfected cells served as control. Values are means ± SEM run in triplicates, and letters indicate significant differences between treatment groups and control.</p

miR-103 is directly involved in IGF-1 stimulated crypt cell proliferation.

<p>IEC-6 cells in 96-well plate were transfected with 50 nM miR-103 mimic before treated with 20 ng/ml IGF-1. The control group was cultured in growth media or treated with IGF-1 alone. Cell proliferation was measured by BrdU incorporation assay. Values are means ± SEM run in triplicates. Letters indicate significant differences between IGF-1 treatment and control groups.</p

Forty-four miRNAs were significantly correlated with mouse crypt cell proliferation upon IGF-1 treatment.

<p>Hierarchical clustering analysis was performed using Euclidian distance. Each row represents relative levels of expression for a significantly regulated single microRNA and each column represents the relative expression level of a single replicate relative to control (<i>P</i><0.01). Colors on the figure represent the scaled fold-change between samples within a gene. Control group was set to 0.</p

miR-103 expression analysis in IGF-1 stimulated mouse small intestinal crypt cells.

<p>(A) microrarray analysis of total miRNA expression in crypt cells after stimulation with IGF-1 for 24 h. The MA plot shows the averaged and background-subtracted fold change on a log2 scale (Y axis) and average expression intensity (X axis) of each miRNA on both channels for Cy-3 labeled control and Cy-5 labeled treated cells and their dye-swaps. Each dot or triangle represents one miRNA probe. Arrow indicates miR-103 probe, which is 44.26% of relative control intensity over the course of 24 h. (B, C) real-time Q-PCR analysis of mature miR-103 (B) and pri-miR-103 expression (C) during 24 h IGF-1 treatment in crypt cells. Data were normalized to snoRNA-202 levels for mature miR-103 and β-actin for pri-miR-103. Values are means ± SEM run in triplicates, letters indicate significant differences between time points.</p

Principal component analysis of plasma metabolites at PD20.

<p>Principal component analysis of plasma metabolite variables from rat pups at PD20 from either a normal (orange) or growth restricted (purple) group. PC1 explains 22.3% of the variation, whereas PC2 explains 18.1%. Inset shows PCA of individual groups colored by amount of iron supplemented. 0 μg (red), 30 μg (green), 150 μg (blue) iron.</p

Colon microbial taxa at PD20.

<p>Significantly altered colon microbial taxa by diet at PD20. In red are taxa that increase, and blue are taxa that decrease in growth-restricted rats. The significance was determined using two-way ANOVA (iron treatment X diet) based on relative abundance data. The significance cut-off is <i>p</i> < 0.05. Disclaimer: in this small-scale pilot study (n = 4–6), multiple comparisons were not adjusted, and therefore results may contain false discoveries. Future confirmation is needed.</p

Principal coordinates analysis (PCoA) of 16s rRNA.

<p>PCoA of unweighted Unifrac distances of 16S rRNA sequences demonstrates clustering along PC1 based on diet (upper half of the figure) (purple represents restricted and orange as normal) for day 20 and 56. Clustering based on iron supplement (lower half of the figure) (0 μg (Control), 30 μg (medium), or 150 μg (high)).</p