Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells (2020)
Figure 4

Figure 4: SCC tumour cells are dependent on USP28 and/or ∆Np63 to maintain a SCC identity

Correlation of gene expression changes upon constitutive transduction of A-431 cells with either shRNA targeting USP28 (sh-USP28#1) or ∆Np63 (sh-∆Np63#2) relative to non-targeting control (sh-NTC). The diagonal line reflects a regression build on a linear model. R: Pearsons correlation coefficient, m: slope of the linear regression model

Venn diagram of differentially up-regulated genes (log2FC>1.5 and q-value<0.05) between sh-∆Np63#2 and sh-USP28#1 relative to non-targeting control (sh-NTC) in A-431 cells. p-values were calculated using a hypergeometric test.

Venn diagram of differentially down-regulated genes (log2FC<-1.5 and q-value<0.05) between sh-∆Np63#2 and sh-USP28#1 relative to sh-NTC in A-431 cells. p-values were calculated using a hypergeometric test.

Gene set enrichment analysis (GSEA) of a gene set of significantly down-regulated genes in sh-∆Np63#2 transfected A-431 cells ("Down-regulated sh-∆NP63", Appendix Table S1). The gene set was analysed in sh-∆Np63#2 (left) and sh-USP28#1-depleted (right) A-431 cells. (N)ES: (normalised) enrichment score

Gene set enrichment analysis (GSEA) of a gene set of significantly down-regulated genes in sh-USP28#1 transfected A-431 cells ("Down-regulated sh-USP28", Appendix Table S1). The gene set was analysed in shUSP28#1 (left) and sh-∆Np63#-depleted (right) A-431 cells. (N)ES: (normalised) enrichment score

GO term analysis of biological processes enriched in sh-∆Np63#2-depleted A-431 cells relative to sh-NTC. Numbers indicate the ranking position of all analysed GO terms based on the significance.

GO term analysis of biological processes enriched in sh-USP28#1-depleted A-431 cells relative to sh-NTC. Numbers indicate the ranking position of all analysed GO terms based on the significance.

Immunoblot of endogenous ΔNp63 and USP28 in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC) or against USP28 and transiently transfected with exogenous ΔNp63. ACTIN served as loading control. Representative western blot from three independent experiments.

Cell growth of A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC) or against USP28 and transiently transfected with exogenous ΔNp63. Total cell number was measured and assessed at indicated time points. Quantitative graph is represented as mean ± SD of three experiments (n=3). p-values were calculated using two-tailed t-test statistical analysis. *p-value < 0.05, **p-value < 0.01

Relative expression of SCC marker genes KRT5, 14 and 19 in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC), two independent shRNA-ΔNp63, two independent shRNA-USP28 or ΔNp63 in shRNA-USP28#1, normalised to ACTIN. Quantitative graph is represented as mean ± SD of three experiments (n=3).

Relative expression of epithelial marker genes KRT10 and GPCR5A in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC), two independent shRNA-ΔNp63 and shRNA-USP28#1, normalised to ACTIN. Quantitative graph is represented as mean ± SD of three experiments (n=3).

Genomic signature of primary human lung SCC samples comprising USP28, ∆Np63, KRT5, KRT14, KRT19, DSG3, MKI67, PCNA and GPRC5A. Samples were sorted dependent on relative USP28 expression (high to low). n=553. Xena UCSC software.

GSEA of consensus squamous cancer marker genes (se Appendix Table S2) in A-431 cells stably transduced with sh-∆Np63#2 or sh-NTC. (N)ES: (normalised) enrichment score

GSEA of consensus squamous cancer marker genes in A-431 cells stably transduced with sh-USP28#1 or sh-NTC. (N)ES: (normalised) enrichment score

Correlation of mRNA expression of consensus squamous cancer marker genes and TP63 in lung SCC and non-transformed lung tissue (Normal). R: Spearmans correlation coefficient. n=836. Gepia software.

Correlation of mRNA expression of consensus squamous cancer marker genes and USP28 in lung SCC and non-transformed lung tissue (Normal). R: Spearmans correlation coefficient. N=836. Gepia software.

Data information: See also Figure EV2 and Appendix Table S3 (Exact P-values and statistical test used).