Extended Data Fig. 5: Initiation of the DDR is impaired in Il22−/− mice.
From: Interleukin-22 protects intestinal stem cells against genotoxic stress
a–c, Il22+/+;Lgr5eGFP and Il22−/−;Lgr5eGFP mice (n = 3 each) were irradiated with 8 Gy (DNA damage) or left untreated (steady-state). LGR5(GFP)+ colon epithelial stem cells were highly purified and gene expression was analysed by RNA-seq. a, Sorting strategy for colonic LGR5+ stem cells. Stem cells were highly purified (>98%) for RNA-seq analysis. b, Modified Venn diagram. All expressed genes with fold change greater than 2 are represented. The bottom half of the diagram represents genes differentially expressed between Il22+/+ and Il22−/− mice at the steady state. ‘Core expression’ contains all genes with detectable transcripts that were not differentially expressed. The top half represents genes differentially expressed 24 h after irradiation in comparison to untreated mice. Of these, 596 genes differed in both genotypes, whereas 638 genes were only regulated in stem cells of Il22+/+ mice and 392 genes were regulated in stem cells of in Il22−/− mice. c, GSEA was performed on all expressed transcripts represented in LGR5+ colon stem cells from Il22+/+ (n = 3) and Il22−/− (n = 3) mice at the steady state. Nominal P value (Kolmogorov–Smirnov) was calculated for ‘Double strand break processing’ (P = 0.01). d, Mice were irradiated with 8 Gy. Expression of DDR effector genes p21 and Puma in colonic epithelial cells was determined by qRT–PCR at the indicated time points after irradiation (n = 3; mean ± s.e.m.). e, List of genes included in the GSEA gene set ‘DDR effector genes’4 (Fig. 2b). f, Mice were irradiated with 8 Gy. Gene expression of Il22bp (also known as Il22ra2) in lamina propria or colon epithelial cells was determined by qRT–PCR at the indicated time points (n = 3; mean ± s.e.m.). g, h, Expression of the indicated genes in colon epithelial cells was determined by qRT–PCR at steady state: MRN complex genes (g; Il22+/+, n = 7; Il22−/−, n = 6; mean ± s.e.m.); DDR transducer genes (h; Il22+/+, n = 10, except Atmin, n = 7 and Parp1, n = 5; Il22−/−, n = 7, except Atmin, n = 4 and Parp1, n = 5; mean ± s.e.m.).
