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. 2020 Sep 28;117(41):25560–25570. doi: 10.1073/pnas.1912772117

Fig. 2.

Fig. 2.

Knockdown of Smad4 results in enrichment of the stem and Paneth cell compartments, which is accompanied by increased global translation and reduced expression of translational repressor 4E-BP1. (A) Quantitative RT-PCR analysis of crypt base columnar stem cell markers in shSmad4 organoids (n = 3). (B) Analysis of small intestinal differentiation markers in shSmad4 organoids (n = 3). (C) Combined staining of in situ hybridization of stem cell marker lgr5 and immunostaining for Paneth cell marker lysozyme in shSmad4 organoids, including quantification of the mRNA particles and lysozyme-positive cells per organoid. *P < 0.05, Student’s t test. (D) l-[35S]-methionine incorporation assay of shSmad4 organoids assessed at day 4 after passaging (n = 3). (E) Clonogenic capacity of single cells that grow out to fully developed organoids, presented as a quantification of the organoid number per 20,000 seeded cells (n = 3). (F) l-[35S]-methionine incorporation assay in Apc−/− Kras+/G12D shSmad4#2 (AKS) and Apc−/− Kras+/G12D shControl (AK) organoids assessed at day 3 after passaging (n = 4). (G) Representative immunoblotting analysis of 4E-BP1 expression in shSmad4 organoids. β-Actin served as a loading control (n = 3). (H) Representative immunoblotting analysis of phospho-4E-BP1 (Thr70), eIF4E, and eIF4G in shSmad4 organoids. Optical density ratios of phospho-4E-BP1/4E-BP1 and 4E-BP1/eIF4E were also calculated (n = 2). (I) m7GTP-agarose pulldown assay in shSmad4 organoids to assess the levels of cap-bound eIF4E, eIF4G, and 4E-BP1 (n = 3). Unbound levels of β-actin 4E-BP1 and eIF4G were detected in supernatants. (J) m7GTP-agarose pulldown assay comparing AKS (shSmad4 #2) to AK organoids (shControl) (n = 3). Data are represented as means ± SEM. Significance (one-way ANOVA) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.