Fig 2: The overexpression of CDX2 in induced gastric organoids(A) A schematic representation of the protocol of gastric organoid differentiation from CDX2-iPSC. The administration of DOX (1 µM) started between days 35 and 40.(B) Immunofluorescence analyses of the MUC5AC and E-cadherin (E-Cad) expression in differentiated gastric organoids at day 37 without DOX treatment. Scale bar, 40 µm.(C) Morphologies of gastric organoids derived from CDX2-iPSC after 1 µM DOX treatment for 10 days from Day 36. The representative data of three independent experiments are shown. Scale bars, 200 µm.(D) An RT-PCR analysis showed the mRNA expression of CDX2 at Day45 with or without DOX treatment for 9 days. The expression of CDX2 was increased in DOX(+) organoids. GAPDH was used as an endogenous control.(E) Immunofluorescence analyses of CDX2 and mCherry in gastric organoids derived from CDX2-iPSC at Day45 with or without DOX treatment for 10 days. Scale bars, 20 µm.

Fig 3: Summary of mutational analysis. (A) Bar plot of number of SNVs for each component of patient samples. (B) Bar plot of indels for each component of patient samples. (C) Proportion of SNVs split into six classes in pyrimidines context. (D) Heatmap of recurrently altered breast cancer genes [6] with at least one mutation found in our study. Numbers on bar plots show overlap coefficient of SNVs (A) and indels (B) between paired patient samples. Note that ML6_Abr has a ploidy estimate of 4, so is copy neutral with LOH of the CDH1 locus. (E) MOBSTER-inferred clonal clusters of ML8_Neg showing two distinct VAF-derived distributions.

Fig 4: Other enteric viruses do not induce EMT in Caco-2 cells.(A) Caco-2 monolayers grown on permeable supports were infected with HAstV-1, HAstV-VA1, T1L, T3SA+ (MOI of 10) or mock-infected and TER (transepithelial electrical resistance) was measured from 0–24 hpi. (B) RNA extracted at 24 hpi from Caco-2 cells infected with HAstV-1, HAstV-VA1, T1L, T3SA+, or mock infected, show only HAstV-1 modulate CDH1, SNAI1, and TWIST1. (C) Infected cells were fixed at 24 hpi and stained for E-cadherin (red), viral capsid (green), and DAPI (blue). (D) Expression of E-cadherin was quantified by immunoblot of HAstV-1, HAstV-VA1, T1L, T3SA+ infected (MOI of 10) or mock infected Caco-2 cell lysates. Bands were then quantified by densitometry and normalized to ß-actin then compared to mock-infection. (E) Na/K ATPase (red) and ezrin (green) localization is disrupted in cells infected with HAstV-1 compared to HAstV-VA1, T1L, T3SA+, and mock infected cells. All error bars indicate standard deviations of two independent experiments performed in triplicate, and asterisks show statistical significance as measured by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. All images are representative of two independent experiments.

Fig 5: Replication is required for HAstV-1 induced EMT.(A) Expression of E-cadherin was quantified by immunoblot of HAstV-1, UV-inactivated HAstV-1, or mock infected Caco-2 cell lysates. Bands were then quantified by densitometry and normalized to ß-actin then compared to mock-infection. (B) RNA extracted at 24 hpi from Caco-2 cells infected with HAstV-1, UV-inactivated HAstV-1, or mock infected show UV-inactivated virus does not modulate CDH1 or SNAI1 regulation as active HAstV-1 does. (C) Na/K ATPase (red) and ezrin (green) localization in Caco-2 cells inoculated with UV-inactivated HAstV-1 shows no difference to mock infected cells. (D) Expression of E-cadherin was quantified by immunoblot of HAstV-1, HAstV-1 + 10µM U0126, or mock infected Caco-2 cell lysates. Bands were then quantified by densitometry and normalized to ß-actin then compared to mock-infection. (E) Na/K ATPase (red) and ezrin (green) localization in Caco-2 cells infected with HAstV-1 in the presence of 10µM U0126 and mock infected cells. All error bars indicate standard deviations of three independent experiments performed in triplicate, and asterisks show statistical significance as measured by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.