Fig. 3
TGF-β1 and Smad4 dependent bulk RNA expression signatures in adenoma organoids. a Adenoma organoids derived from 3 mice per genotype of ApcΔ/ΔSmad4+/+ and ApcΔ/ΔSmad4Δ/Δ were exposed to TGF-β1 (390 pM) (0, 1 and 12 h, eight wells per time point), were then pooled for paired-end bulk RNA-Seq (total of 18 RNA-Seq samples). b Principal components of transcriptional clusters according to genotype and time (vst-transformed). c Venn diagram classifies TGF-β1 and Smad4 dependent down-regulated and upregulated genes. Threshold utilised 1.5 log2FC and adjusted p-value < 0.05. d Volcano plots of differentially expressed genes comparing time and genotype (+/+ = ApcΔ/ΔSmad4+/+, Δ/Δ = ApcΔ/ΔSmad4Δ/Δ). In all volcano plots, genes with -log(p-adj) = 30 are shown. Cut-off values for log2FC (1.5) and adjusted-p-value (1e-3) are marked with dashed lines. ApcΔ/ΔSmad4Δ/Δ DEGs are much reduced in number following TGF-β1 treatment. e Enriched gene modules of DEG using the hallmark gene set enrichment (GSEA with tmod package). Comparisons shown in each column. Red and blue indicate the proportion of genes in a module that are either upregulated or downregulated, respectively. Width of each box relates to its effect size, where lighter, less saturated colours indicate lower significance with p-values. f Heatmap showing time-dependent changes in expression (log2FC) for the 50 genes with the lowest adjusted p-values (Likelihood ration test) and g pathway enrichment for these genes are shown generated using Metascape. h Summary of gene specific expression comparing ApcΔ/ΔSmad4+/+ and ApcΔ/ΔSmad4Δ/Δ with time post TGF-β1 exposure. Statistical comparison performed using the Wald test in Deseq2, followed by Benjamini and Hochberg multiple hypothesis correction.
