Figure 1.
Expression of NEDD9 in human cervical cancer tissue and cells.
(A) Immunohistochemical staining of NEDD9 in squamous carcinoma of the cervix (SCC), adenocarcinoma of the cervix (AC) and normal tissue of the cervix. Original magnification, ×400. (B) Immunohistochemical staining of NEDD9 in metastatic left external iliac lymph nodes of SCC and metastatic right obturator lymph nodes of AC. Original magnification, ×400. (C) Statistical analysis showed NEDD9 overexpression was correlated with FIGO stage, histological grading and metastasis. (D) Expression of NEDD9 mRNA and protein in several cell lines was assessed by quantitative PCR and Western blot analysis. (E) NEDD9 (red) localization and expression were assessed by immunofluorescent analysis in cervical carcinoma SiHa, CaSki, HeLa, C33A cells and human HaCaT keratinocytes. Cells were counter-stained with Hoechest33342 (blue) and visualized at 60× magnification.
Figure 2.
Silencing of NEDD9 resulted in reduced cell migration and invasion.
NEDD9 was knocked down by siRNAs or shRNAs in cervical carcinoma SiHa and HeLa cells. (A) The interference effects were confirmed by quantitative PCR in SiHa and HeLa cells. Expression of NEDD9 was examined by Western blotting in SiHa (B) and HeLa cells (C). Cells were infected with lentiviral vectors encoding shRNA against NEDD9. The results of Transwell assay showed that lentiviral delivery of shRNA targeting NEDD9 resulted in reduced cell invasion (D) and migration (E) in SiHa and HeLa cells. The results of Scratch wound-healing assay further verified that silencing NEDD9 resulted in reduced cell migration (F, G and H). Scale bar, 100 μm. * P < 0.05.
Figure 3.
Overexpression of NEDD9 led to increased cell migration and invasion.
Lentivirus vectors were employed to overexpress NEDD9 in human HaCaT keratinocytes (A). TGFβ (5ng/ml) also increased the expression of NEDD9 in human HaCaT keratinocytes (B). Results of Transwell assay showed that lentiviral delivery of NEDD9 resulted in increased cell invasion (C) and migration (D) in HaCaT cells without TGFβ stimulation. Scale bar, 100 μm.
Figure 4.
NEDD9 regulates tyrosine dephosphorylation of FAK and SRC.
(A and B) Expression of NEDD9-associated oncoproteins, FAK and SRC, was examined by Western blotting. Tyrosine-phosphorylated FAK and SRC, rather than FAK and SRC, were significantly down-regulated while NEDD9 was silenced in SiHa and HeLa cells. (C and D) Moreover, tyrosine-phosphorylated FAK and SRC were significantly upregulated while NEDD9 was exogenously overexpressed in HaCaT cells. (E and F) Results of immunoprecipitation showed that tyrosine-phosphorylated NEDD9, rather than NEDD9, were significantly down-regulated while FAK or SRC was suppressed by FAK inhibitor PF-228 or SRC inhibitor PP2 in SiHa and TGFβ-stimulating HaCaT cells. (G) Results of Transwell assay showed that increased cell invasion and migration by exogenous overexpression of NEDD9 were suppressed by FAK inhibitor PF-228 or SRC inhibitor PP2 in HaCaT cells. Scale bar, 100 μm.
Figure 5.
NEDD9 regulates Vimentin and E-cadherin expression.
Expression of EMT markers, Vimentin and E-cadherin, was examined by Western blotting. (A) Vimentin was down-regulated and E-cadherin was upregulated while NEDD9 was silenced in SiHa and HeLa cells. (B) Vimentin was upregulated and E-cadherin was down-regulated while NEDD9 was overexpressed in HaCaT cells. Results of Transwell assay showed that decreased cell invasion (C) and migration (D) by shRNA of NEDD9 were increased by siRNA against E-cadherin in SiHa and HeLa cells. Scale bar, 100 μm. (E) E-cadherin was upregulated and Vimentin was down-regulated by virtue of FAK inhibitor PF-228 or SRC inhibitor PP2 in HaCaT cells with exogenous NEDD9. (F) Results of immunoprecipitation showed that tyrosine-phosphorylated NEDD9 and total NEDD9 were not significantly regulated while E-cadherin was suppressed by siRNA in HaCaT cells.
Figure 6.
The association between NEDD9 and E6/E7 expression.
(A and B) The result of qPCR and western blots showed that deprivation of E6/E7 by siRNA didn’t change the expression of NEDD9 in HPV-positive SiHa and CaSki cells. (C and D) Interference of NEDD9 didn’t affect the expression of E6/E7 in SiHa and CaSki cells.