Extended Data Fig. 3: Effects of Smad4 restoration on gene expression and signaling.
(a) Overlap between upregulated genes in tumor cells from the pancreas, liver, or lungs at 7 and 14 days after Dox withdrawal. Numbers reflect genes in each category. Complete gene lists are provided in Supplementary Table 1. DEGs = differentially expressed genes. (b) Normalized Smad4 mRNA levels (RNA-seq data; tpm = transcripts per million) in KC-shSmad4 tumor cells from the pancreas, liver, or lungs +/-Dox for 7 or 14 days (n = 5, 5, 6, 5, 6 and 8 independent mice for groups shown, left to right). Box plots represent median +/- upper/lower quartile (box) +/-1.5 x IQR between the first and third quartile. (c) Representative IHC staining for pSMAD2 in KC-shSmad4 tumors ( + Dox) from the pancreas, liver, or lungs. Quantifications (%pSMAD2 cells) are shown on the right (n = 6 independent primary tumor regions or metastatic tumors from 2 mice per organ). (d) Heatmap of differentially expressed genes from the indicated KEGG pathways. Average log2 fold-change and p-values are shown for each organ and timepoint. (e) Heatmap of SMAD4-dependent SMAD2/3 ChIP target genes that are differentially upregulated in liver vs. lung metastases at 7 or 14 days after Dox withdrawal. Average RNA-seq log2 fold-change values are shown for Smad4 ON vs. OFF in each organ and timepoint. The type of genomic binding region is color-annotated on the left. Complete gene lists are provided in Supplementary Table 1. (f) Fraction of upregulated genes in liver and/or lung metastases at 7 or 14 days of Dox withdrawal that are SMAD4-dependent SMAD2/3 ChIP-seq targets. Absolute number of genes per group is shown in parentheses. Statistical analyses were one-way ANOVA (b, c), and Wald test with negative binomial modeling and Benjamini-Hochberg adjustment (d).
