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. Author manuscript; available in PMC: 2022 May 22.
Published in final edited form as: Nat Genet. 2021 May 10;53(6):881–894. doi: 10.1038/s41588-021-00859-2

Extended Data Fig. 10 ∣. SOX2 enhances dsRNA expression by activating endogenous retroviruses transcription.

Extended Data Fig. 10 ∣

a, Pie chart depicts the number of upregulated ERV families in SCPP versus CPP with/without enrichment of gained Klf5 binding sites. b, Bar graph illustrates the enrichment of gained Klf5-binding sites in specific ERV families highlighted in Supplemental Fig. 6a. The background ratio was determined by Klf5 ChIP-seq gained sites/unaffected sites in SCPP vs CPP. P-value calculated by two-sided fisher exact test (Background as control). c, Representative H3K27ac ChIP-seq, Sox2 ChIP-seq, Klf5 ChIP-seq and ATAC-seq tracks, showing increased H3K27ac level, gained Sox2 binding, gained Klf5 binding and increased chromatin accessibility at different RLTR13D6 loci in SCPP versus CPP organoids. d, The relative expression of representative RLTR13D6 ERVs in different organoids was assessed by quantitative RT-PCR. Three independent biological replicates were performed for each condition. Data are shown as mean ± SD, p-value calculated by two-sided t-tests. e, The relative ERV expressions of different gained H3K27ac sites (SCPP vs CPP) across four organoids by quantitative PCR. Three biological replicates were performed for each organoid. The data are shown as mean ± SD, p-value calculated by two-sided t-tests. f, Representative double immunofluorescence images for dsRNA marker J2(dsRNA) and Adar1 in SCPP organoid with shRNA-mediated KLF5 silencing. FITC (green), phalloidin (red) and DAPI (blue) channels were used to detect J2(dsRNA), Adar1, and nucleus, respectively. Scale bar=40 um. (This experiment was repeated once with similar results). g. Quantification of the J2(dsRNA) staining in SCPP organoid with shRNA-mediated Klf5 silencing. The signal of shKlf5 was normalized to cell number and then shNT. Three technical replicates were performed for each condition. The data are shown as mean ± SD, NS: not significant and ****p < 0.0001 as calculated by the two-sided one-way ANOVA test followed by Benjamini-Hochberg correction. h, Quantification of the J2(dsRNA) staining in ESCC cell line TT and TE10 with shRNA-mediated SOX2 or KLF5 knockdown. Data are normalized to cell number and Dox (−) and shown as mean ± SD, NS: not significant and ****p < 0.0001 as calculated by two-sided unpaired t-tests.