Fig 1: Transcriptional regulation of NNMT expression.(a) An NNMT activity assay was performed on UC7 and UC11 cell lines. The activity of NNMT normalized to actin correlated with its expression levels (*P < 0.05, **P < 0.01). (b) Western blotting revealed that expression levels of PAX3 decrease upon silencing of AURKA. Full length blots are shown in Supplementary Fig. S7. (c) Schematic presentation of the NNMT promoter. Two PAX3 binding sites are located within 500 bp upstream of the NNMT transcription start site. (d) Chromatin immunoprecipitation indicates that PAX3 binds to the NNMT promoter and represses its expression in both UC7 (top) and UC11 (bottom) cell lines. Full length gels are shown in Supplementary Fig. S7. (e) Luciferase based promoter analysis of the NNMT promoter with mutations in either of the PAX3 binding sites leads to an increase in luciferase expression in the non-targeted (NT) cells to similar levels as the AURKA KO cells. Mutation at both sites together did not have an additive effect, indicating that both sites are necessary for suppression of NNMT transcription (*P < 0.05).
Fig 2: Knockdown of ST8SIA6-AS1 inhibits tumor growth. (A,B) Tumor volume and weight of nude mice bearing control or ST8SIA6-AS1-silenced NCI-H23 cells. (C) qRT-PCR analysis of ST8SIA6-AS1, miR-125a-3p, and NNMT expression in control or ST8SIA6-AS1-silenced tumor tissues. (D) Western blot testing NNMT protein level in control or ST8SIA6-AS1-silenced tumor tissues. **P < 0.01.
Fig 3: Effects of AURKA A on NNMT expression and cell invasion.(a) Panel of bladder cancer cells shows different levels of AURKA expression in UC5, UC6, UC7, UC 9, UC10 and UC11 cell lines. Note low level of AURKA expression in cultured normal urothelial (NHU) cells. Full length blots are shown in Supplementary Fig. S6. (b) Using a lentiviral shRNA construct AURKA was silenced by ~75% in UC7 and UC11 cells. Full length blots are shown in Supplementary Fig. S6. (c) Heatmap of most significantly up- and down-regulated genes after silencing of AURKA in UC11 cell line. (d) AURKA was silenced using a lentiviral vector. Upon silencing, NNMT is upregulated by 3–4 fold in UC7 and UC11 cell lines. In contrast, overexpression of AURKA in UC5 cells resulted with downregulation of NNMT. Full length blots are shown in Supplementary Fig. S6. (e) Upon silencing of AURKA cell invasion is decreased by ~3 fold in both UC7 and UC11 cells in a matrigel invasion assay (*P < 0.01). (f) A luciferase promoter analysis of NNMT reveals that NNMT’s upregulation is transcriptionally regulated in both cell lines upon silencing of AURKA (*P < 0.05). (g) AURKA KO UC7 and UC11 cells were treated with AURKA rescue vectors and western blot was performed to verify the rescue of AURKA and NNMT expression. Full length blots are shown in Supplementary Fig. S6. (h) Quantitation of cell invasion through matrigel coated invasion chambers in AURKA silenced and rescued UC7 and UC11 cell lines (*P < 0.05). (i) Luciferase based promoter analysis of the NNMT promoter indicated that when AURKA is rescued, luciferase expression is returned to lower than normal levels in both UC7 and UC11cell lines (*P < 0.05). (j) Overexpression of AURKA in UC5 cells which resulted in downregulation of NNMT (see Fig. 1d) increased cell invasion in matrigel coated chambers. (k) NNMT was silenced 80–90% in UC7 and UC11 cell lines using lentiviral shRNA directed against NNMT. A reduction of NNMT expression was observed by western blot. Full length blots are shown in Supplementary Fig. S6. (l) An increase in cell invasion was observed in both cell lines in NNMT shRNA treated cells when compared to NT shRNA (*P < 0.05).
Fig 4: NNMT promoted the cell proliferation and migration of OSCC cells. (a,d) NNMT mRNA and protein expression in several OSCC cell lines. (b,e) Ectopic expression of NNMT in HN6 cells, confirmed using qPCR and Western blot. (c,f) Knockdown expression of NNMT in OSCC3 cells, confirmed using qPCR and Western blot. (g,h) Ectopic expression of NNMT significantly promoted proliferation and migration ability. (i,j) Knockdown expression of NNMT significantly inhibited proliferation and migration ability. ** and *** represented differences were considered statistically significant with p < 0.01 and p < 0.001, respectively.
Fig 5: Correlation between the expression of NNMT and different clinical characteristics. (a,b) NNMT with different lymph node metastasis and WPOI in TCs. (c) Correlation between NNMT expression and the recurrence and metastasis status in TCs. * and ** represent differences that were considered statistically significant with p < 0.05 and p < 0.01, respectively; and ns represents no statistical differences.
Supplier Page from Abcam for Anti-NNMT antibody [OTI3D8]