Figure 4: The distinct patterns of TLR expression in adult IECs are maintained in intestinal organoids.

(A) Images from SI organoids, SI organoids skewed to enrich for Paneth cells, and colon organoids. Paneth cell skewed organoids are smaller, rounder, and contain dark granules. Images are representative of at least 5 independent experiments.

(B) RNA was isolated from SI, Paneth cell skewed, and colon organoids at day 6 after passage and the gene expression in these organoids was assessed using Nanostring. Each point represents an independent experiment. Values shown are the normalized number of transcripts detected. Error bars are mean +/− SEM. * p ≤ 0.5, ** p ≤ .01, *** p ≤ 0.001.

(C)Reporter fluorescence (YFP/GFP or tomato) in live, CD45^-Epcam⁺ total IECs isolated from indicated organoids on day 6 after passage compared to baseline WT fluorescence in these channels. Flow cytometry plots are representative of 3 independent experiments, each including 2–5 mice of each genotype.

(D) TLR expression in SI and colon organoids at day 6 after passage assessed using RNA-sequencing. Values are transcripts per million. Error bars are mean +/− SEM.

(E) Tomato fluorescence in total IECs (live, CD45^- Epcam⁺) or Paneth cells (live, CD45^- Epcam⁺CD24⁺SSC^hi) from SI organoids from WT and TLR5^TOM mice. Flow cytometry plots are representative of 3 independent experiments.

(F) Tomato fluorescence in total IECs (live, CD45^- Epcam⁺) or Paneth cells (live, CD45^- Epcam⁺CD24⁺SSC^hi) from organoids generated from d7 TLR5^TOM mice. WT mouse organoids are shown as a control. Flow cytometry plots are representative of 2 independent experiments.