Fig. 2

TGF-β-Smad3 signaling mediates the effects of aging on the colonic epithelial organoids. a GO terms analysis in the old-mouse organoids compared with the young-mouse organoids. Data are shown as the top 15 upregulated. b Heatmap displaying RNA-seq data of normalized and scaled genes on the young - and old-mouse organoids. The genes were selected from the results of the GO terms analysis and were manually grouped. The relative level of TGF-β expression in RNA-seq in colonic epithelial organoids from young and old mice is shown in the graph. c Relative levels of TGF-β1 mRNA expression in colonic epithelial organoids from young and old mice. Data are reported as the mean ± standard error of the mean (SEM) (n = 7). d Smad2, Smad3, Smad4, and Smad7 mRNA expression in the young- and old-mouse organoids. Data are normalized to the β-actin transcript and are reported as the mean ± SEM (n = 7). e Western blot showing the protein levels of p-Smad2, Smad2, p-Smad3, Smad3, and β-actin in colonic epithelial organoids from young and old mice. The images were cropped from the original blot image in Supplementary Fig. 4a. The blot was cut before hybridization with antibodies. f Western blot analysis of p-JNK, JNK, p-ERK, ERK, p-p38, p38, and β-actin in the young- and old-mouse organoids. The images were cropped from the original blot image in Supplementary Fig. 5a. The blot was cut before hybridization with antibodies. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 by the Mann–Whitney t-test. TGF-β, transforming growth factor β; GO, gene ontology; p-JNK, phospho c-Jun N-termical kinases; JNK, c-Jun N-termical kinases; p-ERK, phospho-extracellular signal-regulated kinases; ERK, extracellular signal-regulated kinases