Figure 4.
p57 overexpression in gastric organoids triggers long-term growth arrest and enforces a secretory phenotype
(A) Scheme of electroporation generating Dox-inducible p57-OE gastric organoids.
(B) Immunolabeling with p57 and Ki67 antibodies in Dox-inducible p57-OE organoids without (−Dox 3d) or with Dox treatment (+Dox 3d). Scale bars, 100 μm.
(C) Experimental scheme of long-term Dox treatment. Stemness maintenance can be assessed by checking organoid regrowth following Dox withdrawal after long-term Dox treatment.
(D) Induction of long-term growth arrest for 3 months in gastric organoids by p57 expression. Merged images of brightfield and mCherry. The orientation of the organoids changed after D70 as the Matrigel was detached. Organoid regrowth was monitored until the second passage after Dox withdrawal. Several bubbles generated during seeding are visible in “D1 after −Dox.” Scale bars, 1 mm.
(E and F) qRT-PCR of chief cell markers (E) and other markers (F) showing log2-fold change (log2 FC) of gene expression in p57-OE organoids (+Dox) compared with no Dox control after 2 weeks of Dox treatment. Tbp was used as a reference gene, and the data were generated from three biological replicates. Statistical significances were determined by unpaired multiple t test. ∗p < 0.05, ∗∗p < 0.01.
(G) Upper images: TEM images of the Dox-inducible p57-OE organoids cultured with (+Dox) or without (−Dox) Dox treatment for 1 week. Yellow arrows indicate mitochondria and purple arrowheads indicate secretory granules. Scale bars, 1 μm. Lower graphs: quantification of the number of mitochondria and granules. Twenty cells of each condition were analyzed. Lines indicate median with 95% confidence interval. Statistical significances were determined by unpaired Welch’s t test. ∗∗p < 0.01, ∗∗∗∗p < 0.0001.
(H) Niche requirements of the p57-OE organoids. p57-OE organoids induced by pretreatment with Dox for 1 week were cultured in each condition together with Dox for 1 month. Organoid growth was examined in complete medium without Dox after replating. CM, complete medium; -E-F, WRNG medium; -W-R, EFNG medium. Scale bars, 1 mm.
(I) p57-OE organoids induced by Dox treatment were cultured in each condition for 1 month. Organoid growth was examined in complete medium without Dox after replating. -E-F, WRNG medium; -E-F+Selu, WRNG medium with selumetinib (100 nM); -E-F+Tra, WRNG medium with trametinib (1 nM). Scale bars, 1 mm.
See also Figure S4.