Figure 6.

5-HT reduces Lgr5+ ISC clonogenicity via HTR4

(A) Expression levels of all 5-HT receptors in RNA-seq data from Lgr5+ and Lgr5- IECs⁷⁵ (GEO: GSE99457). FPKM; Fragments Per Kilobase of transcript per Million mapped reads.

(B) Relative expression of Lgr5 and Htr4 mRNAs in sorted Lgr5-EGFP+ and Lgr5-EGFP- IECs measured using qPCR. Each dot represents an individual mouse (n = 3), (∗p < 0.05, two-tailed unpaired t test). Mean and standard deviation are shown.

(C) Outline of the experiment (left), representative images (middle), and quantification of control and 5-HT-treated organoids (right). A representative of n = 4 experiments is shown; at least 3 technical replicates were analyzed per experiment. Asterisks indicate statistical significance (∗p < 0.05, one-way ANOVA with Tukey’s post hoc test). Mean and standard deviation are shown. Scale bars: 200 μm.

(D) Outline of the experiment (left), representative images (middle), and quantification of Tegaserod-treated (1 μM) organoid formation compared to control organoids (∗p < 0.05, two-tailed unpaired t-test), n = 3 experiments with 3 replicate wells. Mean and standard deviation are shown. Scale bars: 200 μm.

(E) Representative images of single Lgr5+ cells grown for 6 days in indicated conditions. GR, GR113808 (1 μM), 5-HT (2 μM). Quantification of the organoid formation was done using the organoid classifier tool Tellu.⁸⁰n = 3 experiments with 3 replicate wells, except GR+5HT 1μM condition with n = 2 independent experiments. Asterisks indicate statistical significance (∗p < 0.05, one-way ANOVA with Tukey’s post hoc test). Mean and standard deviation are shown. Scale bars: 200 μm.