Extended Data Fig. 10: Analysis of Klf4 and Runx1 depletions in vitro and in vivo. | Nature Cancer

Extended Data Fig. 10: Analysis of Klf4 and Runx1 depletions in vitro and in vivo.

From: SMAD4 induces opposite effects on metastatic growth from pancreatic tumors depending on the organ of residence

Extended Data Fig. 10

(a) Western blot analysis of shRNA efficiency. Actin was used as a loading control. (b) Proliferation assays of KC-shSmad4 cell lines harboring the indicated stable shRNAs +/-Dox and +/-TGFβ in vitro (mean + SEM; n = 3 independent experiments per group). (c) Schematic of experiments to assess early metastatic colonization of KC-shSmad4 cells with TF knockdowns. Created in BioRender. Tsanov, K. (2025) https://BioRender.com/ufvotxg. (d-e) Bioluminescent analysis of livers (d) and lungs (e) harvested 7 days after intracardiac injection of cells expressing the indicated shRNAs. Quantification is shown on the left and representative bioluminescent images on the right (mean + SEM; n = 9, 10 and 10 independent mice per organ for groups shown, left to right). (f-g) Schematic of intrasplenic (f) and tail vein (g) experiments with KC-shSmad4 cells with TF knockdowns. Created in BioRender. Tsanov, K. (2025) https://BioRender.com/ur1sduv and https://BioRender.com/hxar0yr. (h) Quantifications of baseline liver metastasis burden (% tumor area) after intrasplenic injections of KC-shSmad4 cells under continuous Dox administration (Smad4-OFF) (mean + SEM; n = 9, 7 and 9 independent mice per group, respectively). Livers were harvested 64-66 days after injection. (i) Quantifications of baseline lung metastasis burden (bioluminescent signal) after tail vein injections of KC-shSmad4 cells under continuous Dox administration (Smad4-OFF) (mean + SEM; n = 7, 8 and 8 independent mice per group, respectively). Lungs were harvested 57 days after injection. (j) Model of the organ-specific interplay between SMAD4 and KLF4/RUNX1-associated chromatin states. In the liver, active KLF4 allows SMAD4 to act at genes that promote cytostasis, thus necessitating the inactivation of Smad4. In the lungs, more speculatively, active RUNX1 allows SMAD4 to act at pro-fibrotic genes unopposed by its cytostatic program. Partially created in BioRender. Tsanov, K. (2025) https://BioRender.com/74y5yim.

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