Supplementary Figure 3: CircPan3 knockout impairs the stemness of Lgr5-GFP⁺ ISCs.

a, Confirmation of intronic sequences responsible for circPan3 formation. b, Minigene assay for circPan3 detection in circPan3 silenced Lgr5-GFP⁺ ISCs infected with lentiviruses carrying the indicated constructs. Black arrowhead denotes circPan3. c, Scheme for circPan3 knockout mice. Intronic sequences (#2) were deleted through CRISPR/Cas9 approach. d, circPan3^−/− mice were identified by agarose gel electrophoresis. e, circPan3^−/− in Lgr5-GFP⁺ ISCs was detected by PCR. Black arrowheads denote primers for linear Pan3 detection and gray arrowheads denote primers for circPan3. f, g, CircPan3 and Pan3 expression levels in circPan3^−/− mice were detected by Northern blot (f) and Western blot (g). h, Expression levels of the indicated circRNAs in circPan3^−/− and circPan3^+/+ Lgr5-GFP⁺ ISCs were examined by real-time PCR. n = 4 independent experiments. i, Pan3 expression levels in Lgr5^GFPcircPan3^−/− mice were detected by Western blot. j, FACS analysis of Lgr5-GFP⁺ ISCs in small intestines of Lgr5^GFPcircPan3^+/+ and Lgr5^GFPcircPan3^−/− mice. Numbers indicated the percent of Lgr5-GFP⁺ ISCs in the gate, and were shown as means ± SD. n = 6 for each group. k, mRNA expression levels of the indicated ISC markers were examined by real-time PCR. n = 5 for each group. l, Western blot for Pan3 detection in LR^lacZcircPan3^+/+ and LR^lacZcircPan3^–/– mice. m, Representative β-galactosidase images from LR^lacZcircPan3^+/+ and LR^lacZcircPan3^–/– intestines (n = 6 per group). n, Schematic representation of intestinal regeneration. o, Scheme for generation of circPan3^CTG mice. ATG codon of circPan3 peptide was mutated to CTG. p, PCR products from circPan3^CTG mice were used for DNA sequencing to confirm the mutation of ATG. q, Lgr5^GFPcircPan3^CTG/CTG mice were used for Lgr5-GFP⁺ ISCs observation by confocal microscopy. Scale bars, 50 μm. n = 200 fields pooled across 6 mice were used for Lgr5-GFP⁺ ISC detection, and data are shown as mean ± s.d. ns, not significant, by one-tailed Student’s t-test. r, Pan3 expression in indicated mice was detected by Western blot. Data represent at least three independent experiments.