Extended Data Fig. 9: Gpx2 regulates colonocyte stemness and tumorigenesis.

a, Multiplicity of tumours emerged in mini-colons treated with the indicated compound upon oncogenic induction. ***P = 0.0001 (day 5), <0.0001 (all other conditions) (two-way ANOVA and Sidak’s multiple comparisons test; n = 6 mini-colons for each condition). b, Brightfield images of colon organoids treated with the indicated compound after tumorigenic recombination. Images correspond to 3 days after induction. Scale bar, 200 μm. c, Metabolic activity (measured using resazurin) of organoids cultured in the indicated conditions and times after oncogenic recombination. No significant differences (two-way ANOVA and Sidak’s multiple comparisons test; n = 4 cultures for each condition). d, qRT-PCR based quantitation of Gpx2 mRNA in the indicated cell lines. ***P < 0.0001 (one-way ANOVA and Tukey’s multiple comparisons test; n = 6, 4, and 3 organoid cultures for parental, shGpx2 #1, and the rest of the lines, respectively). e, Brightfield images of non-induced colon organoids of the indicated genotypes after 3 days of culture. Scale bar, 200 μm. f, Metabolic activity (measured using resazurin) of non-induced colon organoids of the indicated genotypes at the indicated times. No significant differences (two-way ANOVA and Sidak’s multiple comparisons test; n = 6 cultures for each condition). g, Brightfield images of mini-colons of the indicated genotypes after tumorigenic recombination. Images correspond to 6 days after induction. Scale bar, 75 μm. h, Brightfield images of colon organoids of the indicated genotypes after tumorigenic recombination. Images correspond to 6 days after induction. Scale bar, 200 μm. i, Volcano plot showing the differentially expressed genes upon Gpx2 knockdown in non-transformed colon cells. j, Bubble plot showing the main enriched functional terms in the differentially expressed genes upon Gpx2 knockdown in non-transformed colon cells. Significant terms are highlighted in either blue (downmodulated) or red (upmodulated) (one-sided Fisher’s exact test, adjusted P values). k, Enrichment of the indicated hallmark signatures from the MSigDB in the indicated cell lines. **P = 0.0013; ***P = 0.0008 (Wnt), 0.0003 (EMT, before recombination), <0.0001 (all other conditions); NS, not significant (one-way ANOVA and Tukey’s multiple comparisons test; n = 3 cultures for each condition). l, Colony assay images of non-induced colon organoids of the indicated genotypes after 3 days of culture in the indicated media conditions. Scale bar, 200 μm. m, Clonogenic capacity of non-induced colon organoids of the indicated genotypes after 3 days of culture in the indicated media conditions. *P = 0.0219; **P = 0.0012 (EN, shGpx2 #3), 0.0036 (BMGF, shGpx2 #1); ***P < 0.0001 (two-way ANOVA and Dunnet’s multiple comparisons test; n = 3 cultures for each condition). n, Brightfield images of Gpx2 knockdown mini-colons that had undergone the indicated pre-treatment before tumorigenic recombination. Images correspond to 7 days after tumour induction. Scale bar, 75 μm. In a, c, d, f, k, and m, each point represents one well of organoids and data represent mean ± SEM.

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