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Fig. 1 - Supplemental 1 Regional differences and impacts of microbial density on cargo uptake by lysosome-rich enterocytes (LREs). (A, B) Plots comparing normalized cargo uptake by anterior (50?150 ?m) and posterior (150?250 ?m) LRE regions in 6 dpf germ-free (GF) (A) and conventional (CV) (B) larvae. The anterior LREs took up significantly more mCherry than posterior LREs at 40 min (two-way ANOVA, padj = 0.047, n=9?11) and 60 min PG in GF larvae (two-way ANOVA, padj = 0.0013, n=11). The anterior LREs took up significantly more mCherry than posterior LREs by 60 min post gavage in CV larvae (two-way ANOVA, padj = 0.043, n=9?10). (C) Plot showing % of maximal uptake (saturation) over time from 5 to 60 min post gavage. The anterior LREs of GF larvae (50?150 µm) took up mCherry at a significantly faster rate than CV larvae from 5 to 60 min post gavage (Simple linear regression, p<0.0001, n=9?11). (D) Plot showing % of maximal uptake (saturation) over time. Between 1?5 hr post gavage, mCherry saturation remained constant in the anterior LREs (0?100 ?m) GF larvae (Simple linear regression, p=0.74, n=8?10) but increased in CV larvae (Simple linear regression, p=0.026, n=8?11). The rate of mCherry accumulation was significantly different between GF and CV larvae (Simple linear regression, p=0.0187, n=8?11). (E) Plot showing luminal mCherry fluorescence at 1 and 5 hr post gavage in GF and CV larvae. There was no difference in luminal fluorescence between conditions (two-way ANOVA, padj = 0.17, n=8?11). (F) Plot showing mCherry fluorescence (AU) over time in media containing zebrafish larva microbes or vehicle control. The change in mCherry fluorescence over time was not significantly different between zebrafish microbe and control media (Simple linear regression, p=0.103, n=8?10), showing the zebrafish microbiome did not degrade mCherry. (G) Plot showing Lucifer Yellow (LY) uptake in GF and CV larvae at 3 hr post gavage. There was no difference in LY fluorescence between GF and CV larvae (two-way ANOVA, p=0.98, n=9?11). (H, I) Plots of normalized mCherry fluorescence intensity along the LRE region over time in 6 dpf GF and CV larvae. The LREs in GF and CV larvae took up the same amount of mCherry by 1 hr post gavage when the microbial density was 3×105 CFU/mL (two-way ANOVA, p=0.18, n=10) (H). GF larvae took up significantly more mCherry than CV larvae when the microbial density was 3×106 CFU/mL (two-way ANOVA, p<0.0001, n=10) (I).