Extended Data Fig. 4: Impact of Smad4 restoration on pathways in vitro vs. in vivo, on primary tumor phenotypes, and in GEMMs.
(a) Comparison of gene ontology (GO) analyses of upregulated genes after Dox withdrawal in vitro (x axis) and in vivo in the pancreas, liver or lungs (y axis). See Methods for experimental details. Combined scores, correlation coefficients (ρ) and correlation p-values for the enriched KEGG pathways are shown for each organ. Complete GO lists and combined scores are provided in Supplementary Table 2. (b) Representative immunofluorescence staining for Ki67 in KC-shSmad4 primary tumors +/-Smad4 restoration. mKate2 labels tumor cells. Quantifications are shown on the right (n = 8 independent tumor regions from two mice per group, respectively). Analysis was performed 7 days after Dox withdrawal. (c) Representative immunofluorescence staining for a-SMA in KC-shSmad4 primary tumors +/-Smad4 restoration. mKate2 labels tumor cells. Quantifications are shown on the right (n = 9 and 6 independent tumor regions from two mice per group, respectively). Analysis was performed at experimental endpoint (45 days after Dox withdrawal). (d) Representative immunofluorescence staining for Ki67 in KC-shSmad4 GEMM primary tumors and liver and lung metastases +/-Smad4 restoration. mKate2 labels tumor cells. Quantifications are shown on the right (n = 8, 8, 7, 5, 8 and 8 independent primary tumor regions or metastatic tumors from 3, 4, 4, 3, 3 and 3 mice for groups shown, left to right). Analysis was performed 7 days after Dox withdrawal. (e) Western blot analysis of shRNA efficiency. Smad4-restored cells were pre-treated with TGFb to induce p57 expression. Actin was used as a loading control. (f) Quantifications of baseline metastasis burden (% tumor area) after intrasplenic injections of KC-shSmad4 cells under continuous Dox administration (mean + SEM; n = 9 and 10 independent mice per group, respectively). Livers were harvested 68 days after injection. Statistical analyses were Pearson correlation (a), and unpaired two-sided t-test (b, c, d, f).
