Fig. 3: Atrx loss induces squamous-like plasticity.

a, FACS plots for analysing and sorting EPCAM⁺LY6D⁺ cells from AKP control and AKP Atrx^KO organoids. b, Quantification of the percentage of cells in each EPCAM and LY6D population (n = 3 independent experiments each). c, Quantification of the percentage of LY6D⁺ cells in AKP control and AKP Atrx^KO organoids (n = 3 independent experiments each). P = 0.000019. d, RT–qPCR analysis of squamous cell markers in LY6D^– and LY6D⁺ cells sorted from AKP Atrx^KO organoids (n = 3 independent experiments each). Gene expression was normalized to Actb, and levels relative to LY6D^– cells were calculated using the ΔΔC_t method. e, Representative images of KRT5-stained subcutaneous tumours and lung metastases from mice injected with AKP control or AKP Atrx^KO organoids. f, Quantification of the percentage of KRT5⁺ cells in subcutaneous tumours from mice injected with AKP control or AKP Atrx^KO organoids (n = 5 mice each). g, Quantification of the percentage of KRT5⁺ cells in subcutaneous tumours and lung metastases from mice injected with AKP Atrx^KO organoid cells (n = 5 (subcutaneous) and 6 (lung metastasis) mice). h, Representative images of EPCAM and KRT5 co-immunofluorescence in AKP Atrx^KO subcutaneous tumours. White arrows indicate cells exclusively expressing EPCAM (in EPCAM only panel, magenta), KRT5 (in KRT5 only panel, green) or co-expressing EPCAM and KRT5 (merge). n = 3 biologically independent samples. i, FACS plots for analysing and sorting LY6D⁺ITGA5⁺ cells from AKP control and AKP Atrx^KO organoids. j, Quantification of the percentage of cells in each LY6D and ITGA5 population (n = 3 independent experiments each). k, Schematic of the strategy used to determine the plasticity of different ITGA5 and LY6D expressing cell populations in AKP Atrx^KO organoids. l, Quantification of the percentage of cells in each LY6D and ITGA5 population 9 days after plating. The original plated population is noted on the x axis (n = 4 independent experiments each). m, Representative images of KRT5-stained subcutaneous tumours from mice injected with different LY6D and ITGA5 populations derived from AKP Atrx^KO organoid cells. The population transplanted is indicated above each image (n = 5 mice each). Scale bars, 50 µm. Data are the mean ± s.d. (c,d,f,g). P values were calculated using two-tailed Student’s t-test (c,d) or two-tailed Mann–Whitney test (f,g). Scale bars, 50 µm (e,h,m). The schematic in k was created using BioRender (https://biorender.com).

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