Volume 152, Issue 1, Pages (January 2019)

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Volume 152, Issue 1, Pages 177-184 (January 2019) Dysregulation of Krüppel-like factor 12 in the development of endometrial cancer Ling Ding, Yi Ding, Xiangyi Kong, Jun Wu, Jian Fu, Guijun Yan, Huaijun Zhou Gynecologic Oncology Volume 152, Issue 1, Pages 177-184 (January 2019) DOI: 10.1016/j.ygyno.2018.10.028 Copyright © 2018 Elsevier Inc. Terms and Conditions

Fig. 1 Expression of KLF12 in tissues from endometrial cancer (EC) patients and the association between KLF12 expression and patients' survival rates. (A) Up-regulation of KLF12 mRNA was found in EC tissues (n = 15) compared with normal endometrium (n = 12). ⁎⁎⁎P < 0.001. Student's t-test. (B) Immunohistochemistry (HC) staining of KLF12 expression in EC tissues. KLF12 was positively stained in the cytoplasm and nucleus of the majority of tumor tissues. Normal endometrium and sample of patient 1 were negative for overexpression of KLF12. Samples of patient 2 and patient 3 were evaluated as overexpression of KLF12 (scale bars, 50 μm). (C) Clinical analysis and Kaplan–Meier curves were made to show the survival rate of EC patients with different KLF12 expression level. The cut-off value of KLF12 mRNA expression was 584.49. Gynecologic Oncology 2019 152, 177-184DOI: (10.1016/j.ygyno.2018.10.028) Copyright © 2018 Elsevier Inc. Terms and Conditions

Fig. 2 Detection of KLF12 expression in Ishikawa and HEC-1B cells transfected with Ad-KLF12. (A) Endogenous KLF12 protein expression was detected in Ishikawa and HEC-1B cells using western blotting. (B–C) Relative mRNA and protein expression of KLF12 were measured by RT-PCR and Western blotting in Ishikawa and HEC-1B cells after transfected with Ad-KLF12. Data are expressed as means ± SEM. n = 3, ⁎⁎⁎P < 0.001, one-way ANOVA. (D) The cellular location of expressed KLF12 (Flag-tagged) in Ishikawa cells was analyzed by immunofluorescence staining via confocal microscopy. GFP was used for a cytoplasmic marker, DAPI was used for nuclear staining. Scale bars = 25 μm. Gynecologic Oncology 2019 152, 177-184DOI: (10.1016/j.ygyno.2018.10.028) Copyright © 2018 Elsevier Inc. Terms and Conditions

Fig. 3 KLF12 promoted cell proliferation and migration, and inhibited cell apoptosis. (A) Ishikawa and HEC-1B cells viability were assayed 48 h after transfected with Ad-KLF12 using CCK8. Data are expressed as means ± SEM. ⁎P < 0.05, ⁎⁎P < 0.01, Student's t-test. (B–C) Representative micrographs and quantification colonies were determined by colony formation assay. Data are expressed as means ± SEM. n = 3, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001, one-way ANOVA. (D-E) Phase-contrast microscopy images and statistical results demonstrated Ishikawa and HEC-1B cells' migration gaps were increased after transfected with Ad-KLF12. Data are expressed as means ± SEM. ⁎⁎⁎P < 0.001, Student's t-test. (F) Cell migration was determined using Cell Death Detection Elisa Kit. Data are expressed as means ± SEM. ⁎⁎⁎P < 0.001, Student's t-test. Gynecologic Oncology 2019 152, 177-184DOI: (10.1016/j.ygyno.2018.10.028) Copyright © 2018 Elsevier Inc. Terms and Conditions

Fig. 4 MMP2, MMP9, pAKT S473 and CCND1 are up-regulated in the cells of over-expressing KLF12. (A) Representative immunoblot of multiple proteins from Ishikawa and HEC-1B cells transfected with KLF12, β-Actin was used as loading control. (B) Quantitative graphs of the proteins level. Data are expressed as means ± SEM. n = 4; ⁎P < 0.05, ⁎⁎P < 0.01, Student's t-test. Gynecologic Oncology 2019 152, 177-184DOI: (10.1016/j.ygyno.2018.10.028) Copyright © 2018 Elsevier Inc. Terms and Conditions

Fig. 5 KLF12 promoted tumor growth in vivo. (A) Representative photos of tumorigenesis of mice treated with Ad-KLF12 and Ad-LacZ. Tumors are marked with red arrow. (B) Tumor volumes were evaluated every 3 days, and tumors were harvested and tumor growth curves were made after 27 days. Data are expressed as means ± SD. n = 8; ⁎⁎⁎P < 0.001, Student's t-test. (C) Representative immunoblot of the level of pAKT S473 and CCND1 in the tumors. Gynecologic Oncology 2019 152, 177-184DOI: (10.1016/j.ygyno.2018.10.028) Copyright © 2018 Elsevier Inc. Terms and Conditions