Supplementary Figure 5: circPan3 interacts with Il13ra1 mRNA in Lgr5-GFP⁺ ISCs.

a, The indicated downregulated genes in circPan3^−/− Lgr5-GFP⁺ ISCs were confirmed by real-time PCR. n = 3 PCR primers were listed in Supplementary Table 3. b, The indicated downregulated genes were depleted in Lgr5-GFP⁺ ISCs, followed by reassociation organoid formation assays. Organoid formation ratios were calculated. n = 6 independent experiments. c, d, Lgr5-GFP⁺ ISC cells were isolated from indicated mice, and IL-13Rα1 protein expression on surface and in total was detected by FACS (c) and Western blot (d), respectively. e, Double FISH assays were performed for circPan3 (red) and Il13ra1 mRNA (green) co-localization. f, RNA pulldown assay by incubation of Biotin-labeled circPan3 and Lgr5-GFP⁺ ISC lysates. Eluates were collected for RNA extraction, and Il13ra1 was detected by real-time PCR. Gapdh served as a negative control. n = 4 independent experiments. g, Prediction of stem-loop structures of circPan3. Predictions were based on minimum free energy (MFE) and partition function. Color scales denote confidence of predictions for each base with shades of red indicating strong confidence (http://rna.tbi.univie.ac.at/). HR, hairpin region. h, Lgr5-GFP⁺ ISCs carrying indicated circPan3 mutant were used for detection of circPan3-Il13ra1 mRNA interaction, and Il13ra1 enrichment was detected by real-time PCR. n = 3 independent experiments. i, Il13ra1 mRNA was labeled with Biotin via in vitro transcription, incubated with indicated circPan3 transcript, and subjected into native PAGE for RNA mobility shift assays. j, circPan3-Il13ra1 mRNA interaction detection in Lgr5-GFP⁺ ISCs carrying WT/mutant. Eluates were examined for circPan3 enrichment by real-time PCR. n = 3 independent experiments. k, Biotin-labeled Il13ra1 mRNA was incubated with indicated circPan3 mutations, followed by RNA mobility shift assays. l, Diagram of Il13ra1 mRNA was shown in upper panel and matching sequences and positions were shown in lower panel. In all panels, data are shown as the mean ± s.d. *** P < 0.001, by one-tailed Student’s t-test. For all representative images, at least three independent experiments were performed with similar results.