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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

Fig. 4 ∣. KLF5 is essential for the function of SOX2 in esophageal squamous tumorigenesis.

Fig. 4 ∣

a, Immunoblots (IB) show KLF5 coimmunoprecipitated with SOX2 in human ESCC cell line KYSE70 (top) and mouse SCPP organoid (bottom). (This experiment was repeated twice with similar results.) b, Heatmaps of Klf5 ChIP–seq signals ±2 kb around Sox2 peaks (identified in Fig. 2b) across different organoids. Two independent biological replicates are shown. c, Averaged Klf5 ChIP–seq signal at the gained, unaffected or lost Sox2 binding. Klf5 ChIP–seq signal was averaged from two independent biological replicates. d, Heatmap of averaged Sox2 ChIP–seq signals in gained (top) and lost (bottom) Sox2 binding sites of SCPP organoid upon shRNA-mediated Klf5 knockdown. Two independent biological replicates are shown. e, Venn diagram shows the overlap of Sox2 sites that are gained in SCPP (versus normal) and Sox2 sites that are lost post shRNA-mediated Sox2 silencing in the SCPP organoids. Sites were identified from two independent biological replicates. P < 0.0001 calculated by two-sided Fisher exact test (SCPP shKlf5 unchanged Sox2 binding sites as control). f, Venn diagram shows significant overlap of upregulated genes and downregulated genes (SCPP versus normal) upon shRNA-mediated Klf5 silencing in SCPP organoids. P < 0.0001 calculated by two-sided Fisher exact test (SCPP shKlf5 unchanged genes as control). g, Venn diagrams show significant overlap of downregulated genes upon shRNA-mediated Klf5 knockdown in SCPP organoid and gained Sox2 binding-related genes (top, left), gained Klf5 binding-related genes (top, right), gained H3K27ac sites-related genes (bottom, left) and gained open chromatin sites-related genes (bottom, left) in the comparison of SCPP versus normal. P < 0.0001 calculated by two-sided Fisher exact test (SCPP shKlf5 unchanged genes as control). h, Cell viability of CPP and SCPP organoids was assessed by adenosine triphosphate (ATP) bioluminescence 4 d after control or Klf5 silencing with shRNA. Three independent biological replicates were performed for each organoid. ATP bioluminescence values were normalized to the value of day 0. Data are shown as mean±s.d.; NS, not significant; and P values calculated by two-sided one-way ANOVA followed by Benjamini–Hochberg correction.