Fig 2: 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 3: miR-149-5p mimic suppresses the poly (I:C)-induced IL-6 mRNA and protein and p63 mRNA over-expression in BEAS-2B cells. BEAS-2B cells were cultured in a 24-well plate at 1 × 105 cells per well in BEGM for 24 h. Cells were transfected with the miR-149-5p mimic (5 nM) in BEBM for 24 h and subsequently challenged with poly (I:C) (0.5 μg/mL) in BEBM (ITS+1, 1%) for 24 h. (a) miR-149-5p expression in BEAS-2B cells, n = 3. The Ct value of miR-149-5p was normalised to that of RNU44 (ΔCt). Data are presented relative to the negative control mimic (ΔΔCt). (b) IL-6 mRNA expression in cells, n = 3. (c) IL-6 release from cells assessed using ELISA, n = 4. (d) p63 mRNA expression in cells, n = 3. The Ct value of IL-6 or p63 mRNA was normalised to that of 18S rRNA (ΔCt). Data are presented relative to the negative control (ΔΔCt) as mean ± SEM. (e) Representative immunoblot and (f) densitometric quantification of ΔNp63α protein, n = 3. Values were normalised to those of GAPDH as a loading control. Data are presented relative to the negative control mimic as mean ± SEM. * p ≤ 0.05, compared with the control group, using an unpaired t-test.

Fig 4: Effect of miR-149-5p mimic or antagomir transfection on miR-149-5p, IL-6 and p63 levels in BEAS-2B cells. BEAS-2B cells were cultured in a 24-well plate at 1 × 105 cells/well in BEGM for 24 h. Cells were then transfected with the miR-149-5p mimic or antagomir (5 nM) in BEBM for 24 h (for miRNA and mRNA expressions) or 48 h (for protein levels). miR-149-5p expression in BEAS-2B cells after (a) miR-149-5p mimic transfection or (b) miR-149-5p antagomir transfection. The cycle threshold (Ct) value of miR-149-5p was normalised to that of RNU44 (ΔCt). Data are presented relative to the negative control (ΔΔCt) as mean ± SEM. IL-6 mRNA expression in cells after transfection of (c) mimic or (d) antagomir. p63 mRNA expression in cells after transfection of miR-149-5p (e) mimic or (f) antagomir. The Ct values of IL-6 or p63 mRNA was normalised to that of 18S rRNA (ΔCt). Data are presented relative to the negative control (ΔΔCt) as mean ± SEM. (g) Representative immunoblot and (i) densitometry analysis of ΔNp63α (70 kDa) protein in cells after transfection of miR-149-5p mimic. (h) Representative immunoblot and (j) densitometry analysis of ΔNp63α protein in cells after transfection of miR-149-5p antagomir. Values were normalised to those of GAPDH (37 kDa) as a loading control. Data are presented relative to the negative control as mean ± SEM. * p ≤ 0.05, compared to the control group, using an unpaired t-test, n = 5.

Fig 5: RXRA/B primes SE epigenetic and transcriptomic landscape. A RNA-seq analysis of RXRA, RXRB, and RXRG expression during seven days of RA induction. B Left two panels, phase contrast images of the differentiating hESCs on D2 and D7. Scale bar, 100 μm. Right two panels, immunofluorescence staining of NANOG, KRT18, and TP63 in WT and RXRA/B DKO cells after seven days of differentiation. Scale bar, 100 μm. C Representative GO terms (biological processes) identified from the genes highly expressed in WT or RXRA/B DKO cells after seven days of differentiation. D Heatmap of expression of pluripotency, RA and SE-related genes in WT and RXRA/B DKO cells after two days or seven days of differentiation. E Histogram showing the distribution patterns of RXRA and RXRB peaks in the differentiated cells on D2. F Heatmaps of binding signals of H3K4me3, H3K4me1, H3K27ac, and ATAC-seq at the center of RXRA or RXRB peaks. G Pie chart showing the percentage of RXRA or RXRB peaks located in active enhancers or active promoters. H Scatterplot of differential H3K27ac, H3K4me1, and H3K4me3 peaks in WT versus RXRA/B DKO cells after two days of differentiation. Sites identified as differentially bound with significance (FDR < 0.05) are shown in red. I Metaplots of average H3K27ac, H3K4me1, and H3K4me3 density around RXRA or RXRB peaks in WT and RXRA/B DKO-differentiated cells on D2. J Venn diagram showing the overlap among downregulated genes and the target genes of decreased H3K27ac, H3K4me1, and H3K4me3 signals in differentiated WT versus RXRA/B DKO cells on D2. K Pie chart showing percentage of the overlap genes obtained from (J) with or without RXRA and RXRB binding. L Representative GO terms (biological process) identified from the genes bound by both RXRA and RXRB obtained from (K)