1. ZEB1 promotes epithelial–mesenchymal transition in cervical cancer metastasis 
Jing Ran, Ph.D., Dian-Liang Lin, Ph.D., Rong-Feng Wu, Ph.D., Qiong-Hua Chen, Ph.D., Hui-Ping Huang, M.S., Na-Xuan Qiu, M.S., Song Quan, Ph.D. 
Fertility and Sterility 
Volume 103, Issue 6, Pages e2 (June 2015)
DOI: /j.fertnstert
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
(A–C) Messenger RNA expression levels of Vimentin, E-cadherin, and ZEB1 in SCC tissue from FIGO clinical stage (CIN: n = 10; IB1: n = 10; IB2: n = 10; IIA1: n = 10; IIA2: n = 10; IIB: n = 10) were detected by RT-PCR and normalized to GAPDH expression. (D) The RVE in SCC tissue according to FIGO clinical stage. Expression of ZEB1 and RVE were higher in each stage of SCC than in CINIII tissue. There was no significant difference between IB1 stage (tumor size <4 cm, no vaginal metastasis) and IB2 stage (tumor size >4 cm, no vaginal metastasis). There was no significant difference between IB1 stage (tumor size <4 cm, no vaginal metastasis) and IIA1 stage (tumor size <4 cm, with vaginal metastasis). Additionally, there were significant differences between IIA1 stage (tumor size <4 cm, with vaginal metastasis) and IIA2 stage (tumor size >4 cm, with vaginal metastasis); at the same time, there were significant differences between IIB stage (with parametrial invasion) and IB1, IB2, IIA1, and IIA2 stage (no parametrial invasion). ∗P<.05, ∗∗P<.01, ∗∗∗P<.001, as determined by analysis of variance with Tukey's post hoc test.
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3. Figure 2
Immunostaining of ZEB1, E-cadherin, and Vimentin in cervical SCC tissues. Negative staining of ZEB1 was found in noncancerous cervical epithelia and stromal tissues. Staining of vimentin was absent in the normal cervical squamous epithelia, but was seen in stromal tissues. At the same time, strong expression of E-cadherin was observed in the normal cervical squamous epithelia, but weak expression was seen in the underlying stromal tissues relatively. With the development of cervical cancer, the expression levels of ZEB1 and Vimentin were increased in SCC tissues, especially concentrated at the invasive tumor front and tumor cells. However, the expression of E-cadherin disappeared absolutely in tissues of tumor.
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4. Figure 3
The expression of ZEB1 significantly correlates with E-cadherin and Vimentin in four cervical cancer cell lines. (A–C) Quantitative RT-PCR analysis of the expression of (A) vimentin, (B) E-cadherin, and (C) ZEB1 in four cervical cancer cell lines. (D) The RVE in cervical cancer cell lines. Results are from three independent experiments and are shown as mean ± SEM. ∗P<.05, ∗∗P<.01, ∗∗∗P<.001 (Mann–Whitney U test).
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5. Figure 4
Effects of ZEB1 silencing in Caski-shZEB1 cell and Caski-shCtrl cell. (A) Caski-shZEB1 cells and Caski-shCtrl cells were counted and 1000 cells were plated in 96-well plates. MTS assay was used to detect caski-shZEB1 cell growth from 1-5 days. The spectrometric absorbance of the samples at 490 nm was measured on a microplate reader. The result suggested that from fourth day, the cell growth was significantly slower less than Caski-shCtrl cells (P <.05), especially on the fifth day (P <.01). Results are from three independent experiments in five replicates and shown as mean ± SEM. (B) Wound healing assay was performed on caski-shZEB1 cells and Caski-shCtrl cells (4×105, 6-cm plate). The “wounded” areas were photographed by Nikon Eclipse 50i fluorescent microscope at various time points (0h, 24h, or 48h). One representative experiment was shown, scale bar = 50 μm. (C) Quantitative analysis of wound-induced migration assay from part B. ∗The results were presented as mean ± SEM of three experiments done in duplicate and differences with P<.05 on the Student's t test were considered statistically significant. (D) Western blot of ZEB1, E-cadherin, and Vimentin for Caski cell transfected with ZEB1 knockdown or control oligos. GAPDH was used as a loading control. E-cadherin protein was increased and mesenchymal markers Vimentin protein was decreased in the Caski-shZEB1 cells than in Caski-shCtrl cells. The result is a representative result of two independent experiments.
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6. Supplemental Figure 1
Expression of ZEB1 mRNA and protein in caski-shZEB1 and caski-shCtrl. (A) A stable ZEB1 knock-out caski cell was screened after incubating with 200 ug/ml of G418 for 10 days. The green fluorescence protein was detected in the Nikon Eclipse 50i fluorescent microscope (Tokyo, Japan). (B) Relative mRNA expression of ZEB1 in caski-shZEB1 and caski-shCtrl cell were detected by real-time RT-PCR and normalized to GAPDH expression. (C) Relative expression of ZEB1 protein in caski-shZEB1 and caski-shCtrl cell, and the expression of ZEB1 protein in caski-shZEB1 was dramatic decreased than it in caski-shCtrl. Data were presented as means ± SEM, ∗∗P<0.01.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions