Journal: Frontiers in immunology

Article Title: A novel ΔNp63-dependent immune mechanism improves prognosis of HPV-related head and neck cancer.

doi: 10.3389/fimmu.2023.1264093

Figure Lengend Snippet: FIGURE 2 DNp63 regulates migration, invasion and response to cisplatin in HPV-positive head and neck cancer cells. (A) Spheroids of SCC90 cells transfected with scrambled or DNp63 siRNA. (B) Size of the central SCC90 spheroid area. Data is represented as individual replicates with mean connected (N=3). ANOVA and Bonferonni test: * p<0.05; ** p<0.01; *** p<0.001. (C) Size of the SCC90 spheroid outer cell rim area upon siRNA-mediated DNp63 downregulation. Data is represented as scatter plots with bars and mean +/- SEM (N=3). ANOVA and Bonferonni test: *** p<0.001. (D) Quantification of the migration of SCC90 and SCC47 cells upon DNp63 downregulation and upregulation, respectively (micrographs are shown in Supplementary Figures S2E, H). Data is represented as scatter plots with bars and mean +/- SEM (N≥3). Student t-test: * p<0.05. (E) Quantification of invasion of SCC90 and SCC47 cells upon DNp63 downregulation and upregulation, respectively (micrographs are shown in Supplementary Figures S2E, H). Data is represented as scatter plots with bars and mean +/- SEM (N≥3). Student t-test: * p<0.05. (F) Resazurin cell viability assay of SCC90 spheroids upon DNp63 inhibition. Data is represented as scatter plots with bars and mean +/- SEM (N=3). ANOVA and Bonferonni post-test: *** p<0.001. (G, H) Western blot analysis of DNp63, p53 (G) and HPV16 E6 (H) expression in siDNp63-transfected SCC90 cells. (I) Western blot analysis of DNp63, p53 and cleaved caspase 3 (Cas3*) upon downregulation of DNp63 in SCC90 cells and treatment with cisplatin (IC50 = 2.8 mM; IC75 = 6.7 mM). p53 and Cas3* signals were quantified with respect to the actin loading control and normalized to non-treated siCtrl SCC90 cells or siCtrl SCC90 cells treated with the IC75 of cisplatin, respectively (quantification results are shown). (J) Western blot analysis of p63, p53 and cleaved caspase 3 (Cas3*) expression upon overexpression of DNp63 in SCC47 cells and treatment with cisplatin (IC50 = 2.7 mM; IC75 = 4.0 mM). p53 and Cas3* signals were quantified with respect to the actin loading control and normalized to non-treated siCtrl SCC90 cells or siCtrl SCC90 cells treated with the IC75 of cisplatin, respectively (quantification results are shown).

Article Snippet: DNp63 and DKK3 downregulation was achieved by transfecting 15 nM of DNp63 siRNA (Eurogentec; sense strand: 5 ’- ACGAGGAGCCGTTCTAATC-3’; antisense strand: 5’-ACCTGG AAAACAATGCCCAGA-3’) and 15 nM of DKK3 siRNA (Santa cruz Biotechnology; sc-41102) to 3E05 SCC90 cells using Lipofectamine RNAiMAX, according to the manufacturer’s instructions.

Techniques: Migration, Transfection, Viability Assay, Inhibition, Western Blot, Expressing, Control, Over Expression

Journal: Frontiers in immunology

Article Title: A novel ΔNp63-dependent immune mechanism improves prognosis of HPV-related head and neck cancer.

doi: 10.3389/fimmu.2023.1264093

Figure Lengend Snippet: FIGURE 3 RNA-seq analysis of siDNp63-transfected SCC90 cells. (A) Enrichment analysis of signalling pathways that are repressed upon siRNA-mediated DNp63 downregulation in SCC90 cells. (B) Enrichment analysis of signalling pathways that are activated upon siRNA-mediated DNp63 downregulation in SCC90 cells. (C, D) Top-100 downregulated (C) and upregulated (D) genes in siDNp63-transfected SCC90 cells according to the fold change (FC) and the Log10.adjusted p-value [log10(adp)].

Article Snippet: DNp63 and DKK3 downregulation was achieved by transfecting 15 nM of DNp63 siRNA (Eurogentec; sense strand: 5 ’- ACGAGGAGCCGTTCTAATC-3’; antisense strand: 5’-ACCTGG AAAACAATGCCCAGA-3’) and 15 nM of DKK3 siRNA (Santa cruz Biotechnology; sc-41102) to 3E05 SCC90 cells using Lipofectamine RNAiMAX, according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Transfection

Journal: Frontiers in immunology

Article Title: A novel ΔNp63-dependent immune mechanism improves prognosis of HPV-related head and neck cancer.

doi: 10.3389/fimmu.2023.1264093

Figure Lengend Snippet: FIGURE 5 DNp63 regulates cancer cell uptake by macrophages via DKK3. (A) Analysis of the expression of the DKK3 gene in siDNp63-transfected SCC90 cells. Data is represented as scatter plots with bars and mean +/- SEM (N=4). Two-tailed Student t-test: * p<0.05. (B) Analysis of the expression of the DKK3 protein in the cellular (upper panels) and supernatant (lower panels) fractions of siDNp63-transfected SCC90 cells. (C) Analysis of the expression of the DKK3 protein in the cellular (left panels) and supernatant (right panels) fractions of SCC90 cells transfected with an anti-DKK3 siRNA. (D) Analysis of the in vitro phagocytosis of green-labeled SCC90 cells by red-labeled THP-1 macrophages upon siRNA-mediated DKK3. DAPI, Red cell dye, Green cell dye and merge are shown. White arrowheads indicate green-labeled phagocytosis vesicles. Magnification: X200. A magnification (right panels) of the inset in the merge is shown. (E) Quantification of the proportion (%) of THP-1 macrophages that display green- labeled phagocytosis vesicles. Data is represented as scatter plots with bars and mean +/- SEM (N=4). Two-tailed Student t-test: p=0.029. (F) Quantification of the proportion of THP-1 macrophages that display p65-positive nuclei. THP-1 cells were incubated with DMEM (negative control), 0.1 µg of TNF-a or 0.5 µg of hrDKK4 for 6 h prior to staining (see also Supplementary Figure S6A). Data is represented as scatter plots with bars and mean +/- SEM (N≥3). ANOVA and Tukey post-test: *p<0.05; *** p<0.001. (G) Immunocytofluorescence analysis of the expression of p65 in THP-1 macrophages incubated with conditioned medium from siCtrl- (upper panels) or siDKK3- (lower panels) transfected SCC90 cells. DAPI, p65 staining and merge are shown. Magnification: X200. A magnification (right panels) of the inset in the merge is shown. White and yellow arrowheads highlight p65 staining in the cytoplasm and the nuclei, respectively. A quantification of the proportion of THP-1 macrophages with p65-positive nuclei is plotted in the graph. Data is represented as mean scatter plots with bars and +/- SEM (N=3). Two-tailed Student t-test: * p<0.05. (H) Western blot analysis of the expression of total and phosphorylated (pS473) AKT (left panels), and of total and phosphorylated (pS32) IkB-a in whole protein extracts from THP-1 cells incubated with rhDKK3 for 30 min and 2 h. AKT, AKT-pS473, IkB-a and IkB-a-pS32 signals were quantified with respect to the actin loading control and normalized to THP-1 macrophages incubated with DMEM (quantifications are shown). (I) Western blot analysis of CKAP4 expression in siCKAP4-transfected THP-1 macrophages. CKAP4 signals were quantified with respect to the actin loading control and normalized to siCtrl-transfected THP-1 macrophages (quantifications are shown). Shown blots are representative examples of three independent experiments. (J) A quantification of the proportion of siCtrl- and siCKAP4-transfected THP-1 macrophages with p65-positive nuclei upon incubation with 0.5 µg of rhDKK3 for 6 h. Data is represented as scatter plots with bars and mean +/- SEM (N=4). Two-tailed Student t-test: * p<0.05.

Article Snippet: DNp63 and DKK3 downregulation was achieved by transfecting 15 nM of DNp63 siRNA (Eurogentec; sense strand: 5 ’- ACGAGGAGCCGTTCTAATC-3’; antisense strand: 5’-ACCTGG AAAACAATGCCCAGA-3’) and 15 nM of DKK3 siRNA (Santa cruz Biotechnology; sc-41102) to 3E05 SCC90 cells using Lipofectamine RNAiMAX, according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Two Tailed Test, In Vitro, Labeling, Incubation, Negative Control, Staining, Western Blot, Control