Knockdown of prohibitin expression promotes glucose metabolism in eutopic endometrial stromal cells from women with endometriosis Xiaochen Qi, Yanxin.

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Knockdown of prohibitin expression promotes glucose metabolism in eutopic endometrial stromal cells from women with endometriosis Xiaochen Qi, Yanxin Zhang, Hui Ji, Xiadi Wu, Fuxin Wang, Manxin Xie, Li Shu, Shiwen Jiang, Yundong Mao, Yugui Cui, Jiayin Liu Reproductive BioMedicine Online Volume 29, Issue 6, Pages 761-770 (December 2014) DOI: 10.1016/j.rbmo.2014.09.004 Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Glucose consumption and lactate production in cultures of primary stromal cells isolated from eutopic endometrium with stromal endometrial cells from women with endometriosis (EU), women without endometriosis (EN) or ovarian-endometrioma tissues (EC). After transfer to fresh medium, medium were collected at 12, 24 and 36 h, respectively, for testing (A) glucose consumption and (B) lactate production. Data represented as mean ± SEM, relative to normal controls (endometrial stromal cells isolated from the endometrium in women without endometriosis). *P < 0.05, **P < 0.001. Reproductive BioMedicine Online 2014 29, 761-770DOI: (10.1016/j.rbmo.2014.09.004) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Real-time polymerase chain reaction data for genes related to glucose metabolism. (A) Glucose transporters, GLUT1 and GLUT3, were found down-regulated in stromal endometrial cells isolated from endometrioma tissues. (B) HIF1α and its target gene PDK1 showed up-regulated in stromal cells from endometrioma tissues, whereas PDHA1 and PDHB were down-regulated. (C) Some glycolytic enzymes, including HK1, PKM and LDHA, are aberrantly expressed in endometrial stromal cells from endometrioma tissues at mRNA level. (D) The mRNA expression of SDHA, SDHB, SDHC, SDHD (four units of SDH enzyme) were decreased in stromal cells rom endometrioma tissues, although for SDHC this was only significant when compared with endometrial stromal cells form women with endometriosis. Data represented as mean ± SEM. *P < 0.05, **P < 0.001. Reproductive BioMedicine Online 2014 29, 761-770DOI: (10.1016/j.rbmo.2014.09.004) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Prohibitin (PHB) mRNA expression levels in stromal cells and its sub-cellular localization in T-hESC cell line. (A) PHB was expressed at a low mRNA level in endometrial stromal cells from endometrioma tissues compared with endometrial cells in women with endometriosis and those without. Data represented as mean ± SEM. *P < 0.05, **P < 0.001. (B) Images obtained with Zeiss LSM 700 confocal microscopy. PHB (FITC/green) co-localized with the Mito-tracker Red. Nuclei were stained with Hoechst33252 (blue). Scale bar = 10 µm. (C) Western blots showing nuclear (nuc), cytoplasmic (cyto) and mitochondrial (mito) proteins extracted from T-hESC cells. Cyto* represented cytoplasmic protein excluding mitochondria. The extracts were immunoblotted for PHB; Lamin A/C, a nuclear antigen; β-tubulin is used to access the purity of the mitoplast preparation. Reproductive BioMedicine Online 2014 29, 761-770DOI: (10.1016/j.rbmo.2014.09.004) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Evaluation of glucose consumption and lactate production after knocking down prohibitin (PHB) expression. (A) For quantitative polymerase chain reaction and immunoblot identification of transfection efficiency, PHB mRNA and protein levels decreased by more than 50% 24 and 48 h after transfection, respectively. PHB mRNA data was represented as mean ± SEM, which was from experiment cells from nine paired different individuals. *P < 0.05. (B) At 24 and 36 h time point 24 h after transfection, knockdown of PHB in endometrial stromal cells in women with endometriosis increased glucose consumption. (C) Knockdown of PHB did not affect lactate production. Results were from experiments using cells from at least 4 women. siRNA-Ctl, negative control; siRNA-PHB, siRNA against PHB; *P < 0.05; NS, non-significant. Reproductive BioMedicine Online 2014 29, 761-770DOI: (10.1016/j.rbmo.2014.09.004) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Real-time polymerase chain reaction data for genes related to glucose metabolism after knocking down prohibitin expression. Knockdown of prohibitin in stromal endometrial cells rom women with endometriosis caused increased expression of PKM, ALDOC, LDHA, PDHA1 and PDHB at a ratio of 1.42, 1.23, 1.26, 1.26 and 1.19, respectively, compared with negative controls. HIF1α, ENO1α and ALDOA were not affected. Results were from experiment cells from 9 different women. *P < 0.05 (Supplementary Figure 1). Reproductive BioMedicine Online 2014 29, 761-770DOI: (10.1016/j.rbmo.2014.09.004) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions