ZEB2 mediates multiple pathways regulating cell proliferation, migration, invasion, and apoptosis in glioma
Authors: Xiaoli Yu
Hao Long
Zhiyong Li
Luxiong Fang
Weiren Luo
Ye Song
Songtao Qi
Weiyi Fang
Yuping Peng
Yan Chen
Yan Zhen
Ying Zhou
Zhen Liu
Hao Wang
Chunping Mai
Aibing Wu
Journal: PLoS ONE (2012)
Discipline: Multidisciplinary
Keywords: ZEB2; glioma; cell proliferation; migration; invasion
Affiliations: Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China. 

Abstract:  Background: The aim of the present study was to analyze the expression of Zinc finger E-box Binding homeobox 2 (ZEB2) in glioma and to explore the molecular mechanisms of ZEB2 that regulate cell proliferation, migration, invasion, and apoptosis. Methodology/principal findings: Expression of ZEB2 in 90 clinicopathologically characterized glioma patients was analyzed by immunohistochemistry. Furthermore, siRNA targeting ZEB2 was transfected into U251 and U87 glioma cell lines in vitro and proliferation, migration, invasion, and apoptosis were examined separately by MTT assay, Transwell chamber assay, flow cytometry, and western blot. Results: The expression level of ZEB2 protein was significantly increased in glioma tissues compared to normal brain tissues (P&lt;0.001). In addition, high levels of ZEB2 protein were positively correlated with pathology grade classification (P = 0.024) of glioma patients. Knockdown of ZEB2 by siRNA suppressed cell proliferation, migration and invasion, as well as induced cell apoptosis in glioma cells. Furthermore, ZEB2 downregulation was accompanied by decreased expression of CDK4/6, Cyclin D1, Cyclin E, E2F1, and c-myc, while p15 and p21 were upregulated. Lowered expression of ZEB2 enhanced E-cadherin levels but also inhibited Œ≤-Catenin, Vimentin, N-cadherin, and Snail expression. Several apoptosis-related regulators such as Caspase-3, Caspase-6, Caspase-9, and Cleaved-PARP were activated while PARP was inhibited after ZEB2 siRNA treatment. Conclusion: Overexpression of ZEB2 is an unfavorable factor that may facilitate glioma progression. Knockdown ZEB2 expression by siRNA suppressed cell proliferation, migration, invasion and promoted cell apoptosis in glioma cells. 

Retracted: No

PubPeer comments:
 Concerns about Figure 7B. 1. Most of the bands shown here appear to show sharp vertical transition lines between the lanes, suggesting they have not been run adjacent to each other. This makes it hard to know if the bands were of the same exposure and size. The Beta-actin control bands, however, do not seem to show these transitions. 2. The Caspase-3 bands all look very similar to the Cleaved PARP bands. The exposure might be different, but shapes and background smudges all are unexpectedly similar.  , 
 2020 Expression of Concern. https: journals.plos.org plosone article?id=10.1371 journal.pone.0231386 After publication of this article [1], concerns were raised about the identity of one of the cell lines used in the study, the reporting of sequence information, and the representation of the western blot data. The authors provide the following clarifications and corrections: The U87 glioblastoma cell line used in Figs 2u20137 of this study was purchased from the Chinese Academy of Sciences (Shanghai, China). In 2018 cell line authentication was carried out by a third party company by STR profiling, which confirmed a match with the STR data of the U87 cell line in the databases of ATCC and DSMZ (S1 File). This U87 glioblastoma cell line has been reported elsewhere to be misidentified [2], though likely to be derived from human glioblastoma [3]. A. Apoptosis in U251 and U87 cells was measured by Annexin V-FITC propidium iodide (PI) staining following siZEB2 or control treatment. Early apoptotic cell populations were significantly increased (P 0.01) after siZEB2 transfection. B. Western blot analysis for antiapoptotic PARP and effector Caspase-3, Caspase-6 and Caspase-9. Decreased PARP and increased caspase-3,-6,-9, and Cleaved PARP expression were observed in U251 and U87 cells after ZEB2 downregulation at 72 hr post transfection. u03b2-actin was used as a loading control. Representative u03b2-actin loading controls are shown for PARP and cleaved PARP; corresponding u03b2-actin loading control panels are not available for Caspase-3, Caspase-6, and Caspase-9. Bar graph shows the relative expression of protein among the groups. Data are presented were presented as mean u00b1 SD for three independent experiments. *P 0.05, statistically significant difference. https: doi.org 10.1371 journal.pone.0231386.g001 The control siRNA was designed and synthesized by RiboBio (RiboBio Inc, China) and the sequence information is commercially protected. The antisense strand of the siRNA targeting ZEB2 is 5u2019-UGUUUCAGAACCUGUGUCC-dTdT-3u2019. This was originally reported in the Methods section in 3u2019-5u2019 direction. There is an error in Table 1 which reports a P value of 0.000. The correct P value is P = 0.005. In Fig 4 the U87 sample western blots for Vimentin and u03b2-Catenin were run on the same gel and share the same u03b2-actin loading control. The U87 sample western blots for N-cadherin and Snail were run on the same gel; the N-cadherin and Snail bands shown for the U87-siZEB sample are taken from the same lane, and therefore share the same u03b2-actin loading control. The original uncropped blots underlying Fig 4 are not available. In Fig 7, splicing together of non-adjacent lanes was introduced during manuscript revision in response to review comments. Bands from the same gel were spliced together in order to rearrange the sample order for presentation purposes for Caspase-3, Caspase-6, Caspase-9, PARP, and Cleaved PARP, but the splicing was not clearly marked on the figure. Additionally, the u03b2-actin and the associated labels were not rearranged in accordance with the amended lane order. The u03b2-actin panel shown in the original figure was the loading control for the PARP blot only; all other proteins were blotted on different gels. The internal consistency of the samples was confirmed at the time of the experiments, but the corresponding loading control blots for the other proteins are no longer available, with the exception of cleaved PARP. The western blot for cleaved PARP was carried out in a later experiment, and due to an error during figure preparation, the incorrect blot was used for the cleaved PARP panel in the figure. A revised Fig 7 is provided in which the original lane order is restored for Caspase-3, Caspase-6, Caspase-9 and PARP, which corresponds to the order of the u03b2-actin panel and the sample labels; additionally, in the revised Fig 7, the cleaved PARP panel is replaced with the correct image, and its associated u03b2-actin panel is provided. Corresponding u03b2-actin loading control panels are not available for Caspase-3, Caspase-6, and Caspase-9. The available underlying western blots for Fig 7 are provided here as S2 File. The results of a repeat experiment carried out using a lentiviral shRNA vector to specifically and stably knock down the expression of ZEB2 in U87 cell lines are provided here as S3 File. The PLOS ONE Editors issue this Expression of Concern to alert readers to the errors in the presentation of the western blots in Fig 7 and to the unavailability of both the individual-level data underlying the accompanying bar chart and the correct corresponding loading control images for the Caspase-3, Caspase-6, and Caspase-9 panels. , 
 #1  A revised Fig 7 is provided in which the original lane order is restored for Caspase-3, Caspase-6, Caspase-9 and PARP, which corresponds to the order of the u03b2-actin panel and the sample labels . Could the authors double-check the u03b2-actin control? At right: Fig 6B from [Song et al (2014)] .  ,