Supplementary Figures 1 to 9 from Distinct TP63 Isoform-Driven Transcriptional Signatures Predict Tumor Progression and Clinical Outcomes

Abstract

<p>Supplementary Figure 1. Pathways enriched in PCSSTAp63 and PCSSÎ"Np63 signatures. a-b, -Log10(p-value) of pathways enriched in (a) PCSSTAp63 and (b) PCSSÎ"Np63 signatures. Supplementary Figure 2. Differential expression of Î"Np63 in MCF10-CA1D, HCC95 and Caki-1 cells. a-b, (a) Western blot analysis for Î"Np63 expression in MCF10-CA1D, HCC95 and Caki-1 cells. NHEK-WT and NHEK-shÎ"Np63 were used as positive and negative control for Î"Np63 expression. Actin was used as internal loading control. (b) Western blot analysis for either Î"Np63 knockdown efficiency in MCF10-CA1D and HCC95 treated with either siC or siÎ"Np63, or Î"Np63 overexpression in Caki-1 cells treated with either empty vector (EV) or Î"Np63α-overexpressing plasmid (Î"Np63α). Supplementary Figure 3: Lef1 is a downstream target of Î"Np63 in MCF10-CA1D and HCC95. a-e, (a) Western blot analysis for Î"Np63 and Lef1 expression in MCF10-CA1D and HCC95 cells treated with siÎ"Np63 and Lef1-overexpressing plasmid (Lef1). Actin was used as internal loading control. (b-e) Quantification and fluorescence representative images of EdU (green) incorporation of indicated cell lines. Data are mean {plus minus} SD, n = 3. Asterisks indicate statistical significance, p < 0.05 versus siC or siÎ"Np63, two-tailed t test. Supplementary Figure 4. Lef1 is a downstream target of Î"Np63 in Caki-1. a-j, (a) qRT-PCR for Lef1 in Caki-1 cells expressing either empty vector (EV) or Î"Np63α-overexpressing plasmid (Î"Np63α). Asterisks indicate statistical significance, p < 0.005 two-tailed t test. (b) Western blot analysis for Lef1 in Caki-1 cells expressing either empty vector (EV) or Î"Np63α-overexpressing plasmid (Î"Np63α). Actin was used as internal loading control. (c) Western blot analysis for Lef1 overexpression efficiency in Caki-1 cells expressing either with empty vector (EV) or Î"Np63α-overexpressing plasmid (Î"Np63α). Actin was used as internal loading control. (d-e) Quantification and bright field representative images of anchorage-independent colony formation of indicated cell lines in soft agar assay (per 10X field). Data are mean {plus minus} SD, n = 3. (f) Western blot analysis for Î"Np63 and Lef1 expression in Caki-1 cells treated with Î"Np63α-overexpressing plasmid (Î"Np63α) and siLef1. Actin was used as internal loading control. (g-h) Quantification and fluorescence representative images of EdU (green) incorporation of indicated cell lines. Data are mean {plus minus} SD, n = 3. (i-j) Quantification and bright field representative images of anchorage-independent colony formation of indicated cell lines in soft agar assay (per 10X field). Data are mean {plus minus} SD, n = 3. Asterisks indicate statistical significance, p < 0.0005 versus EV, two-tailed t test. Supplementary Figure 5. Circos plots of the overlap and expression of TAp63 and Î"Np63 signatures with progression signatures of BLCA, KIRC, KIRP and LGG. a-b. (a) Expression profile of genes common to TAp63-/- signature and each cancer progression signature. (b) Expression profile of genes common to Î"Np63-/- signature and each cancer progression signature. Supplementary Figure 6. Validation of TAp63 and Î"Np63 activities in independent KIRC cohorts. a-b, (a) TAp63KIRC and (b) Î"Np63KIRC activities in independent cohort of KIRC across high and low stages. Supplementary Figure 7. Common pathways enriched in corresponding TAp63 and Î"Np63 signatures of BLCA, KIRC, KIRP and LGG. a-f. (a) -Log10(p-value) of pathways enriched in TAp63KIRC, TAp63KIRP and TAp63LGG. (b) -Log10(p-value) of pathways enriched in Î"Np63BLCA, Î"Np63KIRC, Î"Np63KIRP and Î"Np63LGG. ClueGo plots of overlap in corresponding TAp63 and Î"Np63 in (c) BLCA, (d) KIRC, (e) KIRP and (f) LGG progression signatures. Supplementary Figure 8. Î"Np63CTHRC1/COL5A1 in tumor progression of BLCA, KIRC, KIRP and LGG. a-b, (a) Activity and (b) Kaplan-Meier survival of BLCA, KIRC, KIRP and LGG with respect to Î"Np63CTHRC1/COL5A1. Supplementary Figure 9. Survival of BLCA, KIRC, KIRP and LGG cancers with respect to bottom and top quartile gene expression of (a) CTHRC1 and (b) COL5A1.</p>