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. 2021 Mar 31;71(4):665–675. doi: 10.1136/gutjnl-2020-323276

Figure 4.

Figure 4

KLF5 facilitated ERBBs activation and promoted ERBBs dependency. (A) Modified hockey-stick plot representing the rank of Pearson correlation coefficient difference between EGFR (left), ERBB2 (middle), ERBB3 (right) gene dependencies and gene expression in squamous carcinoma cell lines (n=89) and non-squamous carcinoma cell lines (n=367). Pearson correlation was performed between target gene dependency score and gene expression. Correlation coefficient difference was calculated by subtracting Pearson R of squamous carcinoma group from Pearson R of non-squamous carcinoma group. Bigger value represents that a gene whose expression level correlates with gene dependency more strongly in squamous cell carcinoma than non-squamous cell carcinoma. Smaller value represents that a gene whose expression level correlates with gene dependency more strongly in non-squamous cell carcinoma than squamous cell carcinoma. (B) Representative H3K27ac ChIP-Seq tracks showing enhancer elements at EGFR, ERBB2 and ERBB3 locus in OSCC cell lines TE11 and KYSE410. (C) Representative KLF5 ChIP-Seq tracks showing KLF5 binding sites at EGFR, ERBB2 and ERBB3 locus in OSCC cell lines TE11 and KYSE410. (D) Immunoblot analysis of protein levels of phospho-EGFR, total-EGFR, phospho-ERBB2, total-ERBB2, phospho-ERBB3, total-ERBB3 and KLF5 in OSCC cell line TE11 on shRNA-mediated KLF5 knockdown. Immunoblots from one representative experiment (n=2) are shown. (E) mRNA expression of EGFR, ERBB2 and ERBB3 in OSCC cell lines TE9, TE11 and KYSE180 on shRNA-mediated KLF5 knockdown. Data are presented as mean±SD of three technical replicates per group. *P<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 as calculated by the one-way analysis of variance followed by post hoc test with Benjamini-Hochberg correction. NS indicates non-significant. (F) Cell viability of OSCC cell lines TE9, TE11 and KYSE180 was assessed by ATP bioluminescence 5 days after control or KLF5 knockdown with shRNA with/without palbociclib (500 nM) treatment. Two independent biological replicates were performed for each cell line. ATP bioluminescence values were normalised to the value of day 0 and shNT. Data are shown as mean±SD and *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 as calculated by the two-way analysis of variance followed by post hoc test with Benjamini-Hochberg correction. NS, not significant; OSCC, oesophageal squamous cell carcinoma.