Fig 2: Expression and localization of TP63 isoforms and other selected factors associated with CE homeostasis and stratification in absence of Myc. (A, left panel) CE immunostaining of Krt12 (red) and DAPI (blue) on cross-sections of the paraffin-embedded cornea. The Abs utilized were directed to either the N- or the C-terminus of the Krt12 protein. Krt12 accumulation at the CE surface observed in the mutant using either Ab was severely reduced in the CE of the CKO. Bar graphs represent the percentage of Krt12 apical staining along the CE length of cornea cross-sections (mean ± SD, n = 3). (A, right panel) Line-intensity profile plot of Krt12 fluorescence signal across a CE cross-section. The y-axis represents the average intensity of 15 line-intensity scans (31 µm wide) from the basal membrane (BM) to the apical side of the CE taken every 100 µm along the length of each cornea section. The x-axis represents the length of the line scan from the BM to the apical side of the CE (n = 3). (B) CE immunostaining with Ab directed to the epithelial-specific ΔNp63 (n = 4/group). (C) CE immunostaining with the pan (TP) p63 Abs (n = 2 control and 4 CKO). The bar graphs in B and C describe the mean ± SD values of p63 intensities (green fluorescence) over the whole epithelium, normalized by the corresponding nuclear stain (blue fluorescence). (D, left panel) GAPDH-normalized CKO/control chemiluminescence signal ratios (mean ± SD). Number of independent replicates for each protein are indicated on top of the bars. Myc ablation led to a fourfold increase of ΔNp63 and about twofold increases in ETS1 and Notch 1. Krt14 and Krt 12 displayed no substantial differences in two duplicates and were not quantitated. (E) Level of expression of p63 isoforms relative to Gapdh in the wild-type mouse (mean ± SD, n = 3/group). (F) Expression of p63 isoforms and genes involved in the CE differentiation. Data are represented as the fold change in the CKO relative to the control (n = 3/group). ns, not significant (P ≥ 0.05, Student's t-test). *P < 0.05, **P < 0.001. Scale bars: 25 µm.

Fig 3: Abnormal p63 expression changes iCCA transcriptome.Significant differentially expressed genes (DEG) (|log2FC | > log2(1.2) and padj < 0.05) were identified by comparing CCLP1 (CCLP1_sh2 vs CCLP1_scr) and RBE (RBE_over vs RBE_vector) RNA-seq data. A Volcano plot of all detected genes using log2(FoldChange) as x-axis and-log10(p.adjust) as y-axis. Upregulated DEG were shown in red, downregulated DEG were shown in green, while not significant altered genes were shown as grey dots; the p63 gene in each group is marked with a circle. B Significantly enriched terms (pvalue < 0.05) generated by GSEA analysis in CCLP1 group and RBE group respectively. NES > 0 indicates activated terms, NES < 0 indicates suppressed terms. C Venn diagram shows the 1196 overlapped DEG from CCLP1_DEG and RBE_DEG. D GO analysis of overlapped DEG showed significantly enriched GO terms which were classified into three subgroups. E Chord diagrams display enriched GO terms and the expression pattern of associated genes. F PPI network with identified hub genes correlated with p63. G Heatmap of the hub genes expression level. H Correlation between p63 and hub genes was analyzed by Pearson correlation coefficient (r), p < 0.05 and r > 0.40 indicates strong positive correlation. I Survival analysis of hub genes in iCCA patients from NODE dataset (OEP001105), with the mean value as threshold of high-expression group and low-expression group. P-value was calculated using the log-rank test, p < 0.05 was considered as statistically significant.

Fig 4: Identification and quantification of p63 isoforms expressed in iCCA.A Schematic illustration of different p63 isoforms. B qRT-PCR detected the expression of different p63 isoforms in human normal intrahepatic biliary epithelial and intra- and extrahepatic CCA cell lines. Differences were considered significant at p < 0.01. C Western blot assay confirmed the expression of p63 protein in human iCCA cell lines. HaCaT was used as the positive control, and the normal intrahepatic biliary epithelial cell line was used as the negative control.

Fig 5: p63 interaction with YY1 protein.A, B Co-immunoprecipitation (Co-IP) assays showing the interaction between p63 and YY1. Protein extracts (input) were immunoprecipitated with anti-YY1 A or anti-ΔNp63 B antibodies and resolved using SDS-PAGE. Protein–protein interactions were immunoblotted using anti-ΔNp63 A and anti-YY1 antibodies B. C Integrative Genomics Viewer (IGV) image highlighting the p63 binding site overlap with the YY1 binding site, which was obtained from p63 ChIP-seq and YY1 ChIP-seq data.